Inhibitors of the EGF-receptor tyrosine kinase and methods for their use

ABSTRACT

Novel compounds and pharmaceutical compositions useful as EGFR tyrosine kinase inhibitors. Methods of the invention include administration of the EGFR TK inhibitors to treat diseases characterized by enhanced expression of EGF, including cancers, particularly breast cancer. Additionally, a homology model representing the structure of EGFR kinase domain is provided, which model is useful for the rationally design and screening of compounds predicted to bind favorably to EGFR and to inhibit EGFR TK.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.09/273,002, filed Mar. 19, 1999 now U.S. Pat. No. 6,355,678, whichitself claims priority under 35 U.S.C. §119(e) from U.S. ProvisionalApplication 60/090,998, filed Jun. 29, 1998, and from U.S. ProvisionalApplication 60/097,361, filed Aug. 21, 1998.

BACKGROUND OF THE INVENTION

Breast cancer is the most common form of malignancy in women,representing 32% of all new cancer cases and causing 18% of the cancerrelated deaths among women in the USA. Although the majority of patientswith metastatic breast cancer will experience an initial response,survival is only modestly improved with contemporary chemotherapyprograms. Consequently, the development of new anti-breast cancer drugshas become a high priority (Abrams, J. S., et al. M. Cancer 1994, 84,1164).

Human epidermal growth factor (EGF) is a 53 amino acid, single-chainpolypeptide (Mr 6216 daltons), which exerts biologic effects by bindingto a specific cell membrane epidermal growth factor receptor(EGFR/ErbB-1). Many types of cancer cells display enhanced EGFRexpression on their cell surface membranes (Khazaie, K., et al. R. B.Cancer & Metasis Reviews 1993, 12, 255). Enhanced expression of the EGFRon cancer cells has been associated with excessive proliferation andmetastasis (Mendelsohn, J. and Baselga, J. Biologic Therapy of Cancer:Principles & Practice 1995, 607). Examples include breast cancer,prostate cancer, lung cancer, head and neck cancer, bladder cancer,melanoma, and brain tumors (Khazaie, K., et al. R. B. Cancer & MetasisReviews 1993, 12, 255).

In breast cancer, expression of the EGFR is a significant andindependent indicator for recurrence and poor relapse-free survival(Toi, M., et al. European J. Cancer 1991, 27, 977; Chrysogelos, S. A.and Dickson, R. B. Breast Cancer Res. Treat. 1994, 29, 29; Fox, S. B.,et al. Breast Cancer Res. Treat. 1994, 29, 41). Additionally, it hasbeen shown that the EGFR has an essential function for the survival ofhuman breast cancer cells (Uckun, F. M., et al. Clin. Can. Res. 1998, 4,901; Moyer, J. D., et al. Cancer Res. 1997, 57(21), 4838). Therefore,the development of PTK inhibitors which abrogate the enzymatic functionof the EGFR tyrosine kinase has become a focal point in drug discoveryresearch programs aimed at designing more effective treatment strategiesfor metastatic breast cancer (George-Nascimento, et al. Biochemistry1988, 27, 797; Khazaie, K., et al. R. B. Cancer & Metasis Reviews 1993,12, 255; Fry, D. W. and Bridges, A. J. Curr. Opin. BiotechnoL 1995, 6,662; Wakeling, A. E., et al. Breast Cancer Research & Treatment 1996,38, 67).

The primary metabolite of the anti-inflammatory leflunomideN-(4-trifluoromethylphenyl)-5-methylisoxazol-4-carboxamide, has beenidentified as an inhibitor of the EGFR kinase (Parnham, M. J. Exp. OpinInvest. Drugs 1995, 4, 777; Xu, X., et al. Biochem. Pharmacol. 1996, 52,527; Xu, X., et al. J. Biol. Chem. 1995, 270, 12398; Bertolini, G., etal. J. Med. Chem. 1997, 40, 2011; Mattar, T., et al. A. F. E. B. S.Lett. 1993, 334, 161). Despite the identification of this inhibitor ofthe EGFR kinase, however, there is a continuing need for novelanti-cancer drugs. In particular, there is a need for anti-cancer drugswhich are more potent or more selective than existing drugs. There isalso a need for anti-cancer drugs that operate by novel mechanisms, andthus, may be useful against cancers that do not respond to, or havedeveloped resistance to, existing therapies.

SUMMARY OF THE INVENTION

Applicants have discovered compounds that selectively inhibit EGFRtyrosine kinase, without affecting the activity of other PTKs. Arepresentative compound of the invention was also found to inhibit theproliferation and in vitro invasiveness of EGFR positive human breastcancer cells at micromolar concentrations. Thus, the compounds of theinvention are useful for treating cancer (e.g. breast cancer). Thecompounds are also useful as pharmacological tools that can be used tofurther investigate EGFR kinase function, or can be used in competitivebinding assays to help identify other agents that may be useful aspharmaceuticals.

Accordingly, the invention provides a compound of the following formulaI:

where:

-   -   R₁ is (C₁-C₃)alkyl, (₃-C₆)cycloalkyl, phenyl, or NR_(a)R_(b);    -   R₂ is hydroxy, (C₁-C₆)alkoxy, or (C₁-C₆)alkanoyloxy;    -   R₃ is cyano, or (C₁-C₃)alkanoyl;    -   R₄ is hydrogen, or (C₁-C₃)alkyl;    -   R₅ is aryl, or heteroaryl;    -   R₁ and R_(b) are each independently hydrogen, or (C₁-C₃)alkyl;        or R_(a) and R_(b) together with the nitrogen to which they are        attached are pyrrolidino, piperidino, morpholino, or        thiomorpholino;    -   wherein any aryl, or heteroaryl of R₁ and R₅ is optionally        substituted with one or more (e.g., 1, 2, or 3) substituents        independently selected from halo, nitro, cyano, hydroxy,        trifluoromethyl, trifluoromethyl, trifluoromethoxy,        (C₁-C₃)alkoxy, (C₁-C₃)alkyl, (C₁-C₃)alkanoyl, —S(O)₂R_(c), or        NR_(a)R_(b); wherein R_(c) is (C₁-C₃)alkyl, or aryl or a        pharmaceutically acceptable salt thereof.

Prefereably, if R₅ is phenyl, the phenyl is substituted by —S(O)₂R_(c),or is substituted by halo and at least one other substituent.

Preferred compounds of formula I include those of formula II:

where R₁ is (C₁-C₆)alkyl, optionally substituted by 1, 2, or 3substituents selected from the group consisting of halo, hydroxy, amino,(C₁-C₆)alkoxy, and (C₁-C₆)alkanoyloxy; R₂ is hydroxy, (C₁-C₆)alkoxy,(C₁-C₆)alkanoyloxy;R₃ is cyano, alkanoyl; R₆ is amino, hydroxy, cyano,nitro, (C₁-C₆)alkyl, halo(C₁-C₆)alkyl, (C₁-C₆)alkoxy, halo(C₁-C₆)alkoxy,(C₁-C₆)alkanoyl, or (C₁-C₆)alkanoyloxy; and R₇ is H, NH₂, CH₃, OH, CF₃,or halo, preferably, halo is Br or Cl; or a pharmaceutically acceptablesalt thereof.

Particularly compounds of formula I include those of formulae III-VI:

where R₇ is H, NH₂, CH₃, OH, CF₃, or halo. Preferably, halo is Br or Cl.

where R₇ is H, NH₂, CH₃, OH, CF₃, or halo. Preferably, halo is F or Cl.

where R₆ is NH—CH₃ or OCH₃.

where R₂ is —CH₂—CH₂X and X is halo, preferably F; or R₂ is —CH₂CF₃; orR₂ is:

The invention also provides a pharmaceutical composition comprising acompound of formula I; or a pharmaceutically acceptable salt thereof,and a pharmaceutically acceptable carrier.

A particularly useful compound of the invention is LFM 12, having thestructural formula:

The invention also provides a therapeutic method for treating diseasesin which EGFR is overexpressed, particularly cancers (e.g. breastcancer) comprising administering to a mammal in need of such therapy, acompound of the invention, e.g., of formulae I-VI; or a pharmaceuticallyacceptable salt thereof.

The invention also provides a compound for use in medical therapypreferably for use in treating cancer), as well as the use of a compoundof formulae I-VI for the manufacture of a medicament for the treatmentof a pathological condition or symptom in a mammal, such as a human,which is associated cancer (e.g. breast cancer).

The invention also provides a homology model representing the structureof the EGF-R kinase domain and a docking procedure, which are useful torationally design compounds predicted to bind favorably to EGF-R andinhibit EGFR-TK activity. Using this model, leflunomide metaboliteanalogs were designed and found to have potent inhibitory activityagainst EGFR TK (IC₅₀ value of 1.7 μM in EGF-R inhibition assays,killing >99% of human breast cancer cells in vitro by triggeringapoptosis). New potent LFM analogs as active inhibitors of the EGF-Rtyrosine kinase are designed and confirmed using this model.

The invention also provides processes and novel intermediates, describedherein, that are useful for preparing compounds of formulae I, II,IV-VI.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B show a homology model for the EGF-R kinase domain. FIG.1A is a molecular surface model showing the plan as active site. FIG. 1Bis a stereoview of the catalytic site of the EGF-R kinase domain withthe inhibitor binding region represented as a triangular-shaped grid.

FIGS. 2A-2B are photographs showing the homology model of the EGFRkinase domain. FIG. 2A is a ribbon (Cα chain) representation of thehomology model of the EGFR kinase domain and a space filling model ofthe compound LFM-A12 which was docked into the catalytic site. TheN-lobe shown in blue is primarily composed of β-sheets and the C-lobeshown in gray is mostly helical. The hinge region is shown in peach.Prepared using Molscript and Raster3D programs (Bacon, D. J. andAnderson, W. F. J. Molec. Graphics 1988, 6, 219; Kraulis, P. J. Appl.Cryst. 1991, 24, 946; Merritt, E. A. and Murphy, M. E. P. Acta Cryst.1994, D50, 869).

FIG. 2B is a space filling representation of the catalytic site residuesof the EGFR kinase domain. The Cα chain of EGFR is represented as a pinkribbon and the residues comprising the hinge region are shown in blue.One corner of the triangular binding region is located between the T766(peach) and D831 (lavender) residues. The second corner bordering thebinding region is located at R817 shown in green and the third corner isnear the lower left side of the hinge region. A ball and stick model ofthe inhibitor LFM-A12 is shown in multicolor and represents a favorableorientation of this molecule in the kinase active site of EGFR. Preparedusing InsightII program (InsightII, Molecular Simulations Inc. 1996, SanDiego, Calif.).

FIG. 3 shows the superimposed docked positions of the para substitutedLFM analogs at the active site of the EGFR tyrosine kinase. The residuesin the active site are shown in pink. LFM: para-CF₃ substituted compound(active metabolite of leflunomide, green); LFM-A1: para-Br substitutedanalog (blue); LFM-A2: para-Cl substituted analog (red); LFM-A3: para-Fsubstituted LFM analog (white). LFM-A12: para-OCF₃ substituted LFManalog (gold).

FIG. 4 shows the superimposed docked positions of CF₃ substituted LFManalogs in the active site of the EGFR tyro sine kinase. The residues inthe active site are shown in pink. LFM: para-CF₃ substituted compound(active metabolite of leflunomide, green); LFM-A8: meta-CF₃ substitutedanalog (yellow); LFM-A4: ortho-CF₃ substituted analog (red).

FIGS. 5A-5F are anti-phosphotyrosine Western blots showing selectiveinhibition of EGFR tyrosine kinase by LFM-A12. FIG. 5A shows EGFR immunecomplexes from lysates of MDA-MB-231 human breast cancer cells treatedwith LFM or LFM-A12 for 1 hour and then assayed for tyro sine kinaseactivity, as described in Example 5. FIG. 5B shows a lack of inhibitionof IRK immunoprecipitated from HepG2 hepatoma cells in immune complexkinase assays after treatment with LFM or LFM-A12. FIG. 5C shows a lackof inhibition of HCK immunoprecipitated from transfected COS7 cells inimmune complex kinase assays after treatment with LFM or LFM-A12. FIGS.5D-5F show a lack of inhibition of JAK3, JAK1, and BTKimmunoprecipitated from lysates of transfected insect ovary cells.

FIGS. 6A-6B show the docking of LFM-A12 and WHI-P154 into catalyticsites of kinases. FIG. 6A shows the superimposed backbones of thecatalytic site residues of the kinase domain homology models of EGFR(white), PTK (peach) and crystal structure coordinates of HCK (blue),with selected residues at positions A, B, and C. LFM-A12 is shown inmulticolor and represents its docked positionin BTK, which is alsosimilar to its docked position in HCK. The white dotted surface arearepresents the Connolly surface of LFM-A12. FIG. 6B shows superimposedbackbones of the catalytic site residues of the kinase domain homologymodels of EFGR (white), JAK3 (pink) and crystal structure coordinates ofIRK (green), with selected residues at positions A, B, and C. LFM-A12 isshown in multicolor and represents its docked positionin EGFR which isalso similar to its docked position in IRK and JAK3. The white dottedsurface area represents the Connolly surface of LFM-A12.

FIGS. 7A-7B demonstrates anti-invasive activity of LFM-A12 againstMDA-MB-231 human breast cancer cells. Cells were incubated with theindicated concentrations of LFM or LFM-A12 for 24 hours, trypsinized,and placed in Boyden chambers precoated with Matrigel matrix and allowedto migrate for 48 hours. The migrated cells were stained with Hema IIsolution and counted. The data points are the mean values from twoindependent experiments. Untreated breast cancer cells were highlyinvasive in Matrigel-coated Boyden chambers. FIG. 7A is a bar graphshowing the invasion of LFM-A12-treated breast cancer cells through theMatrigel matrix was inhibited in a dose-dependent fashion. The invasionof LFM-treated breast cancer cells was inhibited to a lesser extent. Themean IC₅₀ values were 28.4 μM for LFM-A12 and 97.0 μM for LFM. FIG. 7Bis a series of photographs showing microscopic evidence for thedose-dependent reduction of the numbers of migrated MDA-MB-231 cellsafter treatment with LFM-A12.

FIGS. 8A-8B are graphs showing the cytotoxic activity of LFM and LFM-A12against human breast cancer cells in MTT assays. MDA-MB-231 andMDA-MB-361 cells were treated with LFM (FIG. 8A) or LFM-A12 (FIG. 8B)for 36 hours in 96-well plates and the cytotoxicity was determined bythe MTT assay. The data points represent the mean (±SE) values fromthree independent experiments.

FIGS. 9A-9C are confocal images of LFM-A12—treated breast cancer cells.MDA B-231 cells were treated with LFM-A3 or LFM-A12 at a finalconcentration of 100 μM for 24 hours at 37° C., as described in theExamples. After treatment, cells were processed for immunofluorescenceusing a monoclonal antibody to α-tubulin (green fluorescence). LFM-A12(FIG. 9A) but not LFM-A3 (FIG. 9C)—treated cells showed marked shrinkagewith disruption of microtubules and lost their ability to adhere to thesubstratum. Blue fluorescence represents nuclei stained with TOTO-3. Thecontrol is shown in FIG. 9A FIGS. 10A and 10B diagramatically showregions of LFM-A12 suitable for modificatin to enhance EGFR inhibition.Structural and chemical features of LFM analogs which are proposed toaid binding to the EGFR catalytic site and are described below andillustrated. Binding Mode 1, (FIG. 10A) shows the most likely mode ofbinding of the lead compound LFM-A12 at the EGFR catalytic site. Basedon the modifications of the lead compound, a second mode may also bepossible and this is illustrated in FIG. 10B (Binding Mode 2).

DETAILED DESCRIPTION OF THE INVENTION

The following definitions are used, unless otherwise described: halo isfluoro, chloro, bromo, or iodo. Alkyl, alkoxy, etc. denote both straightand branched groups; but reference to an individual group such as“propyl” embraces only the straight chain group, a branched chain isomersuch as “isopropyl” being specifically referred to.

It will be appreciated by those skilled in the art that compounds of theinvention having a chiral center may exist in and be isolated inoptically active and racemic forms. Some compounds may exhibitpolymorphism. It is to be understood that the present inventionencompasses any racemic, optically-active, polymorphic, orstereoisomeric form, or mixtures thereof, of a compound of theinvention, which possess the useful properties described herein, itbeing well known in the art how to prepare optically active forms (forexample, by resolution of the racemic form by recrystallizationtechniques, by synthesis from optically-active starting materials, bychiral synthesis, or by chromatographic separation using a chiralstationary phase) and how to determine a compounds ability to inhibitEGFR tyrosine kinase using the standard tests described herein, or usingother similar tests which are well known in the art.

Specific and preferred values listed below for substituents and ranges,are for illustration only; they do not exclude other defined values orother values within defined ranges for the substituents.

The term “prodrug moiety” is a substitution group which facilitates useof a compound of the invention, for example by facilitating entry of thedrug into cells or administration of the compound. The prodrug moietymay be cleaved from the compound, for example by cleavage enzymes invivo. Examples of prodrug moieties include phosphate groups, peptidelinkers, and sugars, which moieties can be hydrolized in vivo.

Compounds of the Invention

Specific inhibitors of EGFR tyrosine kinase include those of formula I:

where:

-   -   R₁ is (C₁-C₃)alkyl, (₃-C₆)cycloalkyl, phenyl, or NR_(a)R_(b);    -   R₂ is hydroxy, (C₁-C₃)alkoxy, or (C₁-C₆)alkanoyloxy;    -   R₃ is cyano, or (C₁-C₃)alkanoyl;    -   R₄ is hydrogen, or (C₁-C₃)alkyl;    -   R₅ is aryl, or heteroaryl;    -   R₁ and R_(b) are each independently hydrogen, or (C₁-C₃)alkyl;        or R_(a) and R_(b) together with the nitrogen to which they are        attached are pyrrolidino, piperidino, morpholino, or        thiomorpholino;    -   wherein any aryl, or heteroaryl of R₁ and R₅ is optionally        substituted with one or more (e.g., 1, 2, or 3) substituents        independently selected from halo, nitro, cyano, hydroxy,        trifluoromethyl, trifluoromethyl, trifluoromethoxy,        (C₁-C₃)alkoxy, (C₁-C₃)alkyl, (C₁-C₃)alkanoyl, —S(O)₂R_(c), or        NR_(a)R_(b); wherein R_(c) is (C₁-C₃)alkyl, or aryl or a        pharmaceutically acceptable salt thereof;

Preferred are compounds of formula II, where R₁ is (C₁-C₆)alkyl,optionally substituted by 1, 2, or 3 substituents selected from thegroup consisting of halo, ΔΔΔΔ, (C₁-C₆)alkoxy, R₂ is hydroxy,(C₁-C₆)alkoxy, (C₁-C₆)alkanoyloxy; R₃ is cyano, alkanoyl; R₄ is amino,hydroxy, cyano, nitro, (C₁-C₆)alkyl, halo(C₁-C₆)alkyl, (C₁-C₆)alkoxy,halo(C₁-C₆) alkoxy, (C₁-C₆)alkanoyl, or (C₁-C₆)alkanoyloxy; and R₅ is H,NH₂, CH₃, OH, CF₃, or halo, preferably halo is Br or Cl; or apharmaceutically acceptable salt thereof.

Specifically, (C₁-C₆)alkyl can be methyl, ethyl, propyl, isopropyl,butyl, iso-butyl, sec-butyl, pentyl, 3-pentyl, or hexyl; (C₁-C₆)alkoxycan be methoxy, ethoxy, propoxy, isopropoxy, butoxy, iso-butoxy,sec-butoxy, pentoxy, 3-pentoxy, or hexyloxy; (C₁-C₆)alkanoyl can beacetyl, propanoyl or butanoyl; halo(C₁-C₆)alkyl can be iodomethyl,bromomethyl, chloromethyl, fluoromethyl, trifluoromethyl, 2-chloroethyl,2-fluoroethyl, 2,2,2-trifluoroethyl, or pentafluoroethyl; and(C₂-C₆)alkanoyloxy can be acetoxy, propanoyloxy, butanoyloxy,isobutanoyloxy, pentanoyloxy, or hexanoyloxy. A specific value for R₁ is(C₁-C₆)alkyl, optionally substituted by halo, hydroxy, amino, or(C₁-C₆)alkoxy.

Another specific value for R₁ is (C₁-C₆)alkyl.

A preferred value for R₁ is methyl.

A preferred value for R₁ is hydroxy.

A preferred value for R₃ is cyano.

A preferred value for R₄ is trifluoromethoxy.

A preferred compound is a compound of formula I wherein R₁ is(C₁-C₆)alkyl; R₂ is hydroxy, (C₁-C₆)alkoxy, or (C₁-C₆)alkanoyloxy; R₃ iscyano; and R₄ trifluoromethoxy; or a pharmaceutically acceptable saltthereof.

Another preferred compound is a compound of formula I wherein R₁ ismethyl; R₂ is hydroxy; R₃ is cyano; and R₄ trifluoromethoxy; or apharmaceutically acceptable salt thereof.

Preferred compounds of the invention include novel analogs of LFMdesigned to better fit the EGFR binding pocket and better interact withamino acid residues forming contacts within the pocket. These compoundsof the invention fall within four groups, or types, and are more fullydescribed below in Example 6. These compounds have the followingstructural formulae (III-VI):

where R₅ is H, NH₂, CH₃, OH, CF₃, or halo. Preferably, halo is Br or Cl.

where R₅ is H, NH₂, CH₃, OH, CF₃, or halo. Preferably, halo is F or Cl.

where R₄ is NH—CH₃ or OCH₃.

where R₁ is —CH₂—CH₂X and X is halo, preferably F; or R₁ is —CH₂CF₃; or

Processes for preparing compounds of formulae I-VI are provided asfurther embodiments of the invention and are illustrated by thefollowing procedures and Examples in which the meanings of the genericradicals are as given above unless otherwise qualified. A compound offormula I wherein R₂ is hydroxy, can conveniently be prepared bytreating an intermediate of formula A: with an acid chloride of formula:R₁COCl.

In cases where compounds are sufficiently basic or acidic to form stablenontoxic acid or base salts, administration of the compounds as saltsmay be appropriate. Examples of pharmaceutically acceptable salts areorganic acid addition salts formed with acids which form a physiologicalacceptable anion, for example, tosylate, methanesulfonate, acetate,citrate, malonate, tartarate, succinate, benzoate, ascorbate,ketoglutarate, and glycerophosphate. Suitable inorganic salts may alsobe formed, including hydrochloride, sulfate, nitrate, bicarbonate, andcarbonate salts.

Pharmaceutically acceptable salts may be obtained using standardprocedures well known in the art, for example by reacting a sufficientlybasic compound such as an amine with a suitable acid affording aphysiologically acceptable anion. Alkali metal (for example, sodium,potassium or lithium) or alkaline earth metal (for example calcium)salts of carboxylic acids can also be made.

The compounds of the invention may have attached thereto functionalgroups to provide a prodrug derivative. The prodrug deriviativefacilitates use of the drug in the body, for example, by facilitatingentry into cells. The prodrug derivative may be cleaved or not in theactive compound.

Pharmaceutical Compositions

The compounds of formula I can be formulated as pharmaceuticalcompositions and administered to a mammalian host, such as a humanpatient in a variety of forms adapted to the chosen route ofadministration, i.e., orally or parenterally, by intravenous,intramuscular, topical or subcutaneous routes.

Thus, the present compounds may be systemically administered, e.g.,orally, in combination with a pharmaceutically acceptable vehicle suchas an inert diluent or an assimilable edible carrier. They may beenclosed in hard or soft shell gelatin capsules, may be compressed intotablets, or may be incorporated directly with the food of the patient'sdiet.

For oral therapeutic administration, the active compound may be combinedwith one or more excipients and used in the form of ingestible tablets,buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers,and the like. Such compositions and preparations should contain at least0.1% of active compound. The percentage of the compositions andpreparations may, of course, be varied and may conveniently be betweenabout 2 to about 60% of the weight of a given unit dosage form. Theamount of active compound in such therapeutically useful compositions issuch that an effective dosage level will be obtained.

Conjugation to a Targeting Moiety

The compounds of the invention may be conjugated to a targeting moietyfor targeted delivery to a desired cell. Useful targeting moieties areantibodies or specific ligands that bind to an antigen or ligandreceptor on the specific target cells. For example, the EGFR TKinhibitors of the invention may be targeted to EGFR-expressing cells byconjugation of the inhibitor to an anti-EGFR antibody, or by conjugationto EGF. Other useful targeting moieties include molecules which bind orassociate with the EGFR, including TGF-alpha, Erb2, Erb3, Erb4, orantibodies against these molecules.

To form the conjugates of the invention, a targeting moiety, which isoften a polypeptide molecule, is bound to the compounds of the inventionat reactive sites, including NH₂, SH, CHO, COOH, and the like. Specificlinking agents are used to link the compounds. Preferred linking agentsare chosen according to the reactive site to which the targeting moietyis to be attached.

Methods for selecting an appropriate linking agent and reactive site forattachment of the targeting moiety to the compound of the invention areknown, and are described, for example, in Hermanson, et.al.,Bioconjugate Techniques, Academic Press, 1996; Hermanson, et.al.,Immobilized Affinity Ligand Techniques, Academic Press, 1992; and PierceCatalog and Handbook, 1996, ppT155-T201.

Pharmaceutical Additives

The tablets, troches, pills, capsules, and the like may also contain thefollowing: binders such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, fructose, lactose or aspartame or a flavoring agent such aspeppermint, oil of wintergreen, or cherry flavoring may be added. Whenthe unit dosage form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier, such as a vegetable oilor a polyethylene glycol. Various other materials may be present ascoatings or to otherwise modify the physical form of the solid unitdosage form. For instance, tablets, pills, or capsules may be coatedwith gelatin, wax, shellac or sugar and the like. A syrup or elixir maycontain the active compound, sucrose or fructose as a sweetening agent,methyl and propylparabens as preservatives, a dye and flavoring such ascherry or orange flavor. Of course, any material used in preparing anyunit dosage form should be pharmaceutically acceptable and substantiallynon-toxic in the amounts employed. In addition, the active compound maybe incorporated into sustained-release preparations and devices.

The active compound may also be administered intravenously orintraperitoneally by infusion or injection. Solutions of the activecompound or its salts can be prepared in water, optionally mixed with anontoxic surfactant. Dispersions can also be prepared in glycerol,liquid polyethylene glycols, triacetin, and mixtures thereof and inoils. Under ordinary conditions of storage and use, these preparationscontain a preservative to prevent the growth of microorganisms.

The pharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions or dispersions or sterile powderscomprising the active ingredient which are adapted for theextemporaneous preparation of sterile injectable or infusible solutionsor dispersions, optionally encapsulated in liposomes. In all cases, theultimate dosage form should be sterile, fluid and stable under theconditions of manufacture and storage. The liquid carrier or vehicle canbe a solvent or liquid dispersion medium comprising, for example, water,ethanol, a polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycols, and the like), vegetable oils, nontoxic glycerylesters, and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the formation of liposomes, by themaintenance of the required particle size in the case of dispersions orby the use of surfactants.

The prevention of the action of microorganisms can be brought about byvarious antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, buffers or sodium chloride. Prolonged absorption of theinjectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompound in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfilter sterilization. In the case of sterile powders for the preparationof sterile injectable solutions, the preferred methods of preparationare vacuum drying and the freeze drying techniques, which yield a powderof the active ingredient plus any additional desired ingredient presentin the previously sterile-filtered solutions. For topicaladministration, the present compounds may be applied in pure form, i.e.,when they are liquids. However, it will generally be desireable toadminister them to the skin as compositions or formulations, incombination with a detematology acceptable carrier, which may be a solidor a liquid.

Useful solid carriers include finely divided solids such as talc, clay,microcrystalline cellulose, silica, alumina and the like. Useful liquidcarriers include water, alcohols or glycols or water-alcohol/glycolblends, in which the present compounds can be dissolved or dispersed ateffective levels, optionally with the aid of non-toxic surfactants.Adjuvants such as fragrances and additional antimicrobial agents can beadded to optimize the properties for a given use. The resultant liquidcompositions can be applied from absorbent pads, used to impregnatebandages and other dressings, or sprayed onto the affected area usingpump-type or aerosol sprayers.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts andesters, fatty alcohols, modified celluloses or modified mineralmaterials can also be employed with liquid carriers to form spreadablepastes, gels, ointments, soaps, and the like, for application directlyto the skin of the user.

Examples of useful dermatological compositions which can be used todeliver the compounds of formula I to the skin are known to the art; forexample, see Jacquet et al. (U.S. Pat. No. 4,608,392), Geria (U.S. Pat.No. 4,992,478), Smith et al. (U.S. Pat. No. 4,559,157) and Wortzman(U.S. Pat. No. 4,820,508).

Treatment Methods

The EGFR-tyrosine kinase inhibitors of the invention are useful toinhibit the activity of EGFR-tyrosine kinasae. In particular, thecompounds are usful in the treatment of diseases and pathologicalconditions that involve cells expressing EGFR. Most importantly, theinhibitors of the present invention provide a treatment for selectivelyinhibiting EGFR tyrosine kinase, without significant inhibition of othertyrosine kinases, such as the Src family, Tec family, and Janus familytyrosine kinases.

Many known pathologic conditions involve the EGFR and the activity ofthe EGFR tyrosine kinase. These include cancers such as breast cancer,prostate cancer, lung cancer, brain tumor, bladder cancer, and coloncancer. The EGFR TK inhibitors of the invention are delivered to theEGFR, and particularly to the kinase binding domain of the receptor, inorder to inhibit the kinase activity. The compounds of the invention maybe administered to cancer patients to treat exsisting cancer, or beadministered to those at risk for developing cancer, including genetic,occupational, and nutritional predisposition to cancer.

Additional disorders known to involve EGFR and its TK includeatherosclerosis, disorders of vascular smooth muscle cells, andendothelial cells involved in tumor angiogenesis.

Dosage

Useful dosages of the compounds of formula I can be determined bycomparing their in vitro activity, and in vivo activity in animalmodels. Methods for the extrapolation of effective dosages in mice, andother animals, to humans are known to the art; for example, see U.S.Pat. No. 4,938,949.

Generally, the concentration of the compound(s) of formula I in a liquidcomposition, such as a lotion, will be from about 0.1-25 wt-%,preferably from about 0.5-10 wt-%. The concentration in a semi-solid orsolid composition such as a gel or a powder will be about 0.1-5 wt-%,preferably about 0.5-2.5 wt-%.

The amount of the compound, or an active salt or derivative thereof,required for use in treatment will vary not only with the particularsalt selected but also with the route of administration, the nature ofthe condition being treated and the age and condition of the patient andwill be ultimately at the discretion of the attendant physician orclinician. In general, however, a suitable dose will be in the range offrom about 0.5 to about 100 mg/kg, e.g., from about 10 to about 75 mg/kgof body weight per day, such as 3 to about 50 mg per kilogram bodyweight of the recipient per day, preferably in the range of 6 to 90mg/kg/day, most preferably in the range of 15 to 60 mg/kg/day.

The compound is conveniently administered in unit dosage form; forexample, containing 5 to 1000 mg, conveniently 10 to 750 mg, mostconveniently, 50 to 500 mg of active ingredient per unit dosage form.

Ideally, the active ingredient should be administered to achieve peakplasma concentrations of the active compound of from about 0.5 to about75 μM, preferably, about 1 to 50 μM, most preferably, about 2 to about30 μM. This may be achieved, for example, by the intravenous injectionof a 0.05 to 5% solution of the active ingredient, optionally in saline,or orally administered as a bolus containing about 1-100 mg of theactive ingredient.

Desirable blood levels may be maintained by continuous infusion toprovide about 0.01-5.0 mg/kg/hr or by intermittent infusions containingabout 0.4-15 mg/kg of the active ingredient(s).

The desired dose may conveniently be presented in a single dose or asdivided doses administered at appropriate intervals, for example, astwo, three, four or more sub-doses per day. The sub-dose itself may befurther divided, e.g., into a number of discrete loosely spacedadministrations; such as multiple inhalations from an insufflator or byapplication of a plurality of drops into the eye.

The invention may be better understood with reference to the followingExamples, which are not intended to limit the scope of the invention.

EXAMPLE 1 Crystal Structures of Leflunomide Metabolite and Its Analogs

Small molecule X-ray crystal structures were obtained for 10 of the 15leflunomide metabolite analogs studied herein. The compounds werecrystallized using various solvents by evaporation, vapor diffusion orliquid—liquid diffusion. X-ray data on single crystals were collected ona SMART CCD detector (Bruker Analytical X-ray Systems, Madison, Wis.)using MoKα radiation (λ=0.7107 Å). The space group for each crystal wasdetermined based on systematic absences and intensity statistics. Adirect methods solution provided most of the non-hydrogen atoms from theelectron density map. Several full-matrix least squares/differenceFourier cycles were performed to locate the remaining non-hydrogenatoms. All non-hydrogen atoms were refined with anisotropic thermalparameters. Hydrogen atoms were placed in ideal positions and refined asriding atoms with relative isotropic temperature factors. The structurewas refined using full-matrix least-squares data on F².

Crystal structure calculations were performed using a Silicon GraphicsINDY R4400-SC computer (Silicon Graphics Inc., Mountain View, Calif.) orPentium computer using the SHELXTL V 5.0 (Sheldrick, G. SHELXTL BrukerAnalytical X-ray Systems, Madison, Wis.) suite of programs. The crystaldata, experimental parameters, and refinement statistics are summarizedin Table 1.

TABLE 1 Crystal data, data collection and refinement statistics forleflunomide metabolite analogs.

Compound LFM LFM-A1 LFM-A2 LFM-A7 LFM-A8 X p-CF₃ p-Br p-Cl o-F m-CF₃Empirical formula C₁₂H₉F₃N₂O₂ C₁₁H₉BrN₂O₂ C₁₁H₉ClN₂O₂ C₁₁H₉FN₂O₂C₁₂H₉F₃N₂O Crystal system Triclinic Triclinic Monoclinic MonoclinicTriclinic Space group P-1 P-1 P2₁/n P2₁/c P-1 Cell constants a =9.4862(6)Å A = 4.9906(2)Å a = 13.4297(10)Å a = 8.9641(8)Å a = 5.2176(6)Åb = 11.5511(7)Å B = 9.3735(3)Å b = 3.8073(3)Å b = 14.1215(12)Å b =10.4929(2)Å c = 12.0013(7)Å c = 11.8869(1)Å c = 21.201(2)Å c =8.3270(7)Å c = 12.0757(14)Å α = 96.126(2)° α = 77.394(2)° β = 98.779(2)°β = 101.023(2)° α = 67.719(2)° β = 105.914(1)° β = 86.4042)° β =78.921(2)° γ = 110.571(2)° γ = 88.065(2)° γ = 78.915(2)° Z 4 2 4 4 2Formula weight 270.21 281.11 236.66 220.20 270.21 Reflections 5372 27415021 5017 3660 collected Independent 3715 1836 1849 1788 1967reflections R indices R1 = 0.090 R1 = 0.062 R1 = 0.086 R1 = 0.047 R1 =0.060 (I > 2σ(I)) wR2 = 0.214 wR2 = 0.152 wR2 = 0.205 wR2 = 0.115 wR2 =0.139 Compound LFM-A9 LFM-A10 LFM-A11 LFM-A12 LFM-A13 X m-Br m-Cl m-Fp-OCF₃ 2,5-diBr Empirical formula C₁₁H₉BrN₂O₂ C₁₁H₉ClN₂O₂ C₁₁H₉FN₂O₂C₁₂H₉F₃N₂O₃ C₁₁H₈Br₂N₂O₂ Crystal system Triclinic Triclinic MonoclinicTriclinic Monoclinic Space group P-1 P-1 P2₁/c P-1 P2₁/c Cell constantsa = 5.2782(2)Å A = 5.2955(4)Å a = 4.7724(1)Å a = 4.6460(1)Å a =5.6134(1)Å b = 10.2335(4)Å B = 10.0638(7)Å b = 24.1536(1)Å b =9.0781(3)Å b = 9.9847(3)Å c = 11.5754(4)Å c = 11.2503(8)Å c = 9.1565(2)Åc = 14.6881(1)Å c = 21.5896(2)Å α = 69.792(1)° α = 103.951(2)° β =95.937(1)° α = 94.488(2)° β = 93.639(1)° β = 78.592(1)° β = 102.516(1)°β = 91.658(2)° γ = 75.837(1)° γ = 105.121(2)° γ = 93.682(2)° Z 2 2 4 2 4Formula weight 281.11 236.66 220.20 286.21 360.00 Reflections 3713 34106832 3856 5918 collected Independent 1926 1829 1851 2059 2109reflections R indices R1 = 0.056 R1 = 0.051 R1 = 0.051 R1 = 0.0741 R1 =0.040 (I > 2σ(I)) wR2 = 0.145 WR2 = 0.1277 wR2 = 0.149 wR2 = 0.18 wR2 =0.094

EXAMPLE 2 Construction of a Homology Model for the EGF Receptor KinaseDomain

A homology model for the EGFR kinase domain was constructed based on astructural alignment of the sequence of EGFR (accession # P00533,SWISS-PROT, Univ. of Geneva, Geneva, Switzerland) obtained from Genbank(National Center for Biotechnology Information, Bethesda, Md.) with thesequences of known crystal structures of other protein kinases (kinasedomains of HCK (Sicheri, F., et al. Nature 1997, 385, 602), FGFR(Mohammadi, M., et al. Science 1997, 276, 955), IRK (Hubbard, S. R. TheE. M. B. O. Journal 1997, 16, 5572), and cAPK (Zheng, J., et al. ActaCryst. 1993, D49, 362)).

A multiple sequence alignment of HCK, FGFR, IRK, and cAPK with that ofEGFR was carried out manually, conserving the overall secondarystructure within the family. Once the correspondence between amino acidsin the reference and model sequences were made, the coordinates for thestructurally conserved regions were assigned based on the coordinates ofthe reference proteins. Insertions, deletions and mutations wereincorporated into the template structure to build an initial model. Thefinal model of EGFR was subjected to energy minimization to refine themolecular structure so that any steric strain introduced during themodel-building process could be relieved (Brunger, A. T. X-PLOR 1992,New Haven, Conn.).

The model was screened for unfavorable steric contacts and, ifnecessary, such side chains were remodeled either by using a rotamerlibrary database or by manually rotating the respective side chains. Themodeling was carried out on a Silicon Graphics INDIGO2 computer (SiliconGraphics Inc., Mountain View, Calif.) using the Homology module inINSIGHTII (InsightII, Molecular Simulations Inc. 1996, San Diego,Calif.). The final homology model of the EGFR kinase domain had an rmsdeviation of 0.006 Å from ideal bond lengths and 2.0° from ideal bondangles after energy minimization. The above procedure was used toconstruct the homology model, and the homology model was used, inconjunction with small molecule crystal structures of the leflunomideanalogs, for modeling studies of the EGF-R/LFM complexes.

The modeled EGFR kinase domain has the expected protein kinase fold withthe catalytic site in the center dividing the kinase domain into twolobes. It is composed of a smaller N-terminal lobe connected by aflexible hinge (residues 764 to 773) to a larger C-terminal lobe (SeeFIGS. 1A-1B and 2A-2C). The N-terminal lobe is rich in β-strands, whilethe C-terminal lobe is mostly helical. The catalytic site is defined bytwo β-sheets that form an interface at the cleft between the two lobes.The catalytic site of the EGFR kinase domain displays a remarkablyplanar triangular binding pocket, which can bind the base ring portionof ATP. The sides of this triangle are approximately 15 Å×12 Å×12 Å andthe thickness of the binding pocket is approximately 7 Å, with anestimated volume of approximately 600 Å³. The characteristics of thistriangular region, which binds the base ring portion of ATP, waselucidated using a binding sphere surface calculated by the programSPHGEN in DOCK3.5. (Kuntz, I. D. et al., J. Mol. Biol., 1982:161:269-288). Two sides of the triangle can be visualized as beginning at anapex located between Thr⁷⁶⁶ (peach residue in FIG. 1B) and Asp⁸³¹(lavender residue in FIG. 1B), and extending towards thesolvent-accessible opening of the catalytic site. One side of thetriangle extends from the apex along the hinge region of the catalyticsite (blue residues in FIG. 1B), and a second side extends from the apexto Arg⁸¹⁷ (green residue in FIG. 1B) which is immediately adjacent tothe binding subsites for the sugar and triphosphate groups of ATP. Thehinge region of the binding site is composed of residues 764 to 773(FIG. 2). The third side of the triangle extends along the slot-shapedopening to the catalytic site.

The crystal structures of the HCK/quercetin complex (Sicheri, F., et al.Nature 1997, 385, 602) and two FGFR/inhibitor complexes (Mohammadi, M.,et al. Science 1997, 276, 955) revealed that the reported threeinhibitors of HCK and FGFR bind to the catalytic sites of the respectivetyrosine kinases. When the catalytic sites are superimposed with EGFR,all atoms of the three PTK inhibitors fall within the plane of thetriangle described previously, and each molecule is in close contactwith the superimposed hinge region and Asp⁸³¹ of EGFR. Moreover, theycharacteristically occupy only half of the triangle, near the hingeregion. This molecular fitting feature seems to correlate with tighterbinding and may be an important determinant for effective inhibitorbinding. Similarly, the size and planar shape of the catalytic sitewithin the constructed EGFR kinase domain are likely to contribute toits ability to form energetically favorable interactions with planarmolecules such as the LFM analogs described herein. These considerationsare in agreement with conclusions derived from the structure-activityrelationship analyses of pyrolo- and pyrazoloquinazoline compounds(Palmer, B. D. et al. J. Med. Chem. 1997, 40, 1519) and were thereforeincorporated into the modeling strategy.

While most of the catalytic site residues of the EGFR kinase domain wereconserved relative to other PTKs, we noted a few specific variations.EGFR residues Leu⁶⁹⁴, Val⁷⁰², Lys⁷²¹, and Ala⁷¹⁹ are conserved in EGFR,HCK, FGFR and IRK. Residues Asn⁸¹⁸ and Asp⁸³¹ (opposite to the hinge)are conserved in EGFR, HCK, FGFR, IRK, BTK, JAK1, and JAK3. ResiduesCys⁷⁵¹ and Thr⁸³⁰ are specific for EGFR but vary in BTK (Val, Ser), JAK1(Val, Gly), JAK3 (Val, Ala), IRK (Val, Gly), and HCK (Val, Ala).Residues Thr⁷⁶⁶ and Leu⁷⁶⁸ in the hinge region changes to Met and Leu inIRK, Met and Phe in JAK1, Met and Tyr in JAK3, and to Thr and Tyr inBTK. The right side of the binding pocket (FIGS. 2-4) contains Cys⁷⁷³ inEGFR and is therefore considerably more hydrophobic than thecorresponding residue of PDGFR (Asp), FGFR (Asn), JAK1 (Ser), HCK (Ser),and IRK (Asp). These residue differences provide a basis for designingselective inhibitors of the EGFR kinase domain.

Thus, the catalytic site of the EGFR kinase has a triangular shapedbinding region which contains some specific nonconserved residues. Thisinformation, combined with the homology model developed and describedabove, can be used to design compounds that will possess activity asEGFR kinase inhibitors.

Selectivity for a particular tyrosine kinase can also be achieved byextending the binding area to the other half of the triangle (oppositethe hinge), as has been observed in one of the FGF-R inhibitorstructures (Mohammadi, M., et al. Cell 1996: 86, 577-87) and (Connolly,M. L., Science, 1983, 221: 709-13). The difference in binding affinityis provided by Phe489 which extends from a beta hairpin toward theinhibitor. EGF-R has a shorter and more rigid beta hairpin that FGFRwhich would present some limitations to this strategy. The non-conservedresidue Arg 817 could also provide some binding discrimination if theinhibitor binds nearby. We concluded from our modeling studies that thecatalytic site of the EGF-R has specific features which can beadvantageous for the design of inhibitors. These features include atriangular shaped region which is accessible to an inhibitor. Wehypothesized that molecules fitting the triangular shape of the EGF-Rcatalytic site which can also form favorable contacts with the hingeregion of the binding site will bind more strongly and hence inhibitEGF-R more effectively. In order to elucidate the structure activityrelationships determining the ability of LFM to inhibit the EGF-Rtyrosine kinase as well as test the predictive value of our homologymodel of the EGF-R kinase domain, we have designed and synthesizedanalogs of this compound by systematically replacing the p-CF₃substituent in the phenyl ring. Our modeling calculations were based onthe homology model of the EGF-R kinase domain described above andcrystal structures of the LFM and its analogs. The positions of thecritical residues in the active site of the EGF-R and the dockedpositions of the LFM analogs are shown in FIGS. 2-3. The dockedpositions of the compounds indicate that the molecules maintain a closecontact with the hinge region. In all cases the nitrile nitrogen of theligand was involved in hydrogen bonding with the amide NH of Met. Inmodeling the inhibitory activity of LFM analogs with EGF-R tyrosinekinase, we calculated the binding constants (K_(i) values) based on thebinding interaction between the compounds and the catalytic site of theEGF-R kinase domain.

EXAMPLE 3 Structure-Based Design and Synthesis of LFM Analogs HavingPotent EGFR-Inhibitory Activity

We hypothesized that molecules fitting the triangular shape of the EGFRcatalytic site which can also form favorable contacts with the hingeregion of the binding site would bind more strongly and hence inhibitthe EGFR kinase more effectively. In modeling studies aimed atidentifying LFM analogs with a high likelihood to bind favorably to thecatalytic site of the EGFR kinase domain, we chose to calculate theK_(i) values based on the binding interaction between the inhibitor andEGFR residues. The K_(i) values were calculated for several differentinhibitors and were used to rank the predicted binding strength. Each ofthe small molecule LFM analogs described below was individually modeledinto the catalytic site of the EGFR kinase domain using an advanceddocking procedure. The position of quercetin in the HCK crystalstructure (Sicheri, F., et al. Nature 1997, 385, 602) was used as atemplate to obtain a reasonable starting point for the dockingprocedure. The various docked positions of each LFM analog wasqualitatively evaluated and consequently compared with the IC₅₀ valuesof the compounds in cell-free EGFR kinase inhibition assays. Table 2lists the interaction scores and calculated K_(i) values for LFM and itsanalogs.

TABLE 2 Interaction scores, estimated K_(i) values and measured IC₅₀values for LFM analogs.

Cancer Cell Cytotoxicity IC₅₀ EGFR (μM) Lipo Ludi Ludi^(b) InhibitionMDA- MDA- Compound X HB^(a) Score Score K_(i)(μM) IC₅₀(μM) MB-231 MB-361LFM p-CF₃ 1 522 508 8 5.4 198.9 190.5 LFM-A1 p-Br 1 462 44932 >100 >300 >300 LFM-A2 p-Cl 1 456 443 37 >100 >300 >300 LFM-A3 p-F 1397 362 >100 >100 >300 >300 LFM-A4 o-CF₃ 1 442 354 >100 >100 >300 >300LFM-A5 o-Br 1 424 383 >100 >100 >300 >300 LFM-A6 o-Cl 1 385345 >100 >100 >300 >300 LFM-A7 o-F 1 430 416 69 74.5 >300 >300 LFM-A8m-CF₃ 1 492 465 22 >100 >300 >300 LFM-A9 m-Br 1 367354 >100 >100 >300 >300 LFM-A10 m-Cl 1 391 344 >100 >100 >300 >300LFM-A11 m-F 1 400 387 >100 >100 >300 >300 LFM-A12 p-OCF₃ 1 510 489 131.7 53.4 26.3 LFM-A13 2,5-diBr 1 436 340 >100 >100 >300 >300 LFM-A14none 1 397 367 >100 >100 >300 >300 ^(a)HB = Number of hydrogen bonds.^(b)Ludi K_(i) calculated based on the empirical score function in Ludiprogram(Bohm, H. J. J. Comput. Aided. Mol. Des. 1992, 6, 593; and 36.Bohm, H. J. J. Comput. Aided Mol. Des. 1994, 8, 243). Cell-free EGF Rkinase assays and MTT assays were performed as described in the Methodsand the corresponding IC50 values were calculated from thedose-dependent inhibition curves.Docking Procedure and Evaluation of Protein-Inhibitor Interactions

Fixed docking in the Affinity program within InsightII (InsightII,Molecular Simulations Inc. 1996, San Diego, Calif.) was used for dockingthe LFM analogs to the EGFR tyrosine kinase catalytic site. A triangularbinding region, where the base ring of ATP can bind to EGFR, was definedusing a binding sphere surface calculated by the program SPHGEN inDOCK3.5 (Kuntz, I. D., et al. J. Mol. Biol. 1982, 161, 269). Themodeling calculations used to study the predicted binding of inhibitorsto EGFR were based on the homology model of EGFR described above and thecoordinates of energy-minimized models of the compounds which were usedfor docking. The utility of the inhibitor model coordinates werevalidated by their crystal structures which were obtained later andshowed very similar molecular conformations. Each LFM analog wasinteractively docked into the triangular binding pocket of EGFR based onthe position of quercetin in the HCK/quercetin crystal structure.

The hydrogens on the EGFR were generated and potentials were assigned toboth receptor and ligand prior to the start of the docking procedure.The docking method in the InsightII program uses the CVFF force fieldand a Monte Carlo search strategy to search for and evaluate dockedstructures (InsighII, Molecular Simulations Inc. 1996, San Diego,Calif.). While the coordinates for the bulk of the receptor was keptfixed, the program has the ability to define a radius of residues within5 Å distance from the LFM analog. As the modeling calculationsprogressed, the residues within the defined radius were allowed to shiftand/or rotate to energetically favorable positions to accommodate theligand. Calculations were carried out on an INDIGO2 computer (SiliconGraphics Inc., Mountain View, Calif.) using the CVFF force field in theDiscover program and Monte Carlo search strategy in Affinity (InsighII,Molecular Simulations Inc. 1996, San Diego, Calif.). No solvationprocedures were used. Conjugated gradient minimization was used toconserve CPU time, as the total number of movable atoms was greater than200. Calculations approximating hydrophobic and hydrophilic interactionswere used to determine the ten best docking positions of each LFM analogin the EGFR catalytic site. The various docked positions of each LFManalog was qualitatively evaluated using a score function in the Ludimodule (Bohm, H. J. J. Comput. Aided. Mol. Des. 1992, 6, 593; Bohm, H.J. J. Comput. Aided Mol. Des. 1994, 8, 243) of the program INSIGHTII(InsighII, Molecular Simulations Inc. 1996, San Diego, Calif.) which wasthen used to estimate a binding constant (K_(i)) for each compound inorder to rank their relative binding capabilities and predictedinhibition of EGFR. The calculated K_(i) trends for the LFM analogs werecompared with the trend of the experimentally determined tyrosine kinaseinhibition as well as cytotoxicity IC₅₀ values for the compounds, inorder to elucidate the structure-activity relationships (SAR)determining the potency of LFM analogs.

The docked inhibitors were sandwiched between two regions of mostlyhydrophobic residues. The region above the inhibitor consisted ofresidues Leu⁶⁹⁴, Val⁷⁰², Lys⁷²¹, and Ala⁷¹⁹, whereas the region belowincluded residues Leu⁸²⁰ and Thr⁸³⁰. The positions of the criticalresidues in the active site of the EGFR and the docked positions of theLFM analogs are shown in FIGS. 2-4. Of the possible orientations of theLFM analogs in this binding pocket, the one shown in FIGS. 2-4 had thehighest interaction score and was assumed by all 15 compounds. Thisindicates that the depicted docked positions represent an energeticallyfavored binding mode. In this binding mode the compounds can maintainclose contact with the hinge region on the edge of the inhibitor,residues Leu⁶⁹⁴ and Val⁷⁰² above the inhibitor, and Leu⁸²⁰ and Thr⁸³⁰below. In all cases the nitrile nitrogen of the ligand was involved inhydrogen bonding with the amide NH of Met⁷⁶⁹. In addition thepara-substituted OCF₃ group appeared to form close contacts betweenresidues Thr⁷⁶⁶ and Asp⁸³¹.

From the modeling studies, it was apparent that ortho substitutions onthe phenyl ring of LFM analogs would prevent the molecule from havingclose contact with the hinge region of the receptor. The poorinteraction is reflected by the higher K_(i) values calculated for thisgroup of compounds (LFM-A4-A7; Table 2). The results indicated a trendthat for a smaller substitution at the ortho position, there would beless disruption of close contacts with the receptor. As the substitutedgroup got bulkier, the inhibitor would be pushed further away from thehinge region of the receptor, thereby weakening the contacts. Thecompound containing the smallest ortho-substituted group, LFM-A7 (witho-F) had the best interaction score in this group with a calculatedK_(i) of 69 μM. Docking analysis suggested that LFM-A7 had slightlyincreased contact with EGFR residues relative to the unsubstitutedcompound, LFM-A14, consistent with its lower calculated K_(i) value.

The modeling also revealed that meta substituents, except for m-F, wouldlikely be sandwiched between residues Thr⁷⁶⁶ and Asp⁸³¹. The m-F groupof LFM-A11 was predicted to be located on the opposite side of themolecule relative to the meta-substituted groups of the other 3compounds, and docking showed that the m-F group of LFM-A11 would belocated near Asn⁸¹⁸, instead of the usual binding mode where the metagroup is located between Thr⁷⁶⁶ and Asp⁸³¹, opposite Asn⁸¹⁸. Thecalculated K_(i) values for compounds with m-F, m-Cl and m-Brsubstitutions were greater than 100 μM (Table 2). The results shown inTable 2 indicate that LFM-A8, which has a m-CF₃ substitution, is thebest fitting compound in this group with a calculated K_(i) of 22 μM.While maintaining the close contact with the hinge region, the m-CF₃substituent of LFM-A8 would extend further into a leucine-rich pocket ofthe protein (beyond Thr⁷⁶⁶ and Asp⁸³¹) and gain more hydrophobiccontact. Consequently, LFM-A8 was predicted to bind to the catalyticsite of the EGFR kinase domain better than the other meta-substitutedLFM analogs.

The modeling also revealed that the LFM analogs with para substitutionswould have the greatest potential for inhibition of the EGFR, with p-CF₃and p-OCF₃ being the most promising (calculated K_(i)˜8-13 μM), followedby p-Cl and p-Br, (calculated K_(i)˜30-40 μM), and p-F (calculatedK_(i)>100 μM) (Table 2). The best docked positions of the five parasubstituted compounds showed a binding pattern similar to that of themeta substituted compounds except for a slight shift of the phenyl ringstoward Thr⁷⁶⁶ (FIG. 3). The para substituted compounds would maintain aclose contact with the hinge region of the EGFR kinase domain and bestabilized by additional contact area between the para substituents andthe residues deep inside the binding site. The CF₃ and OCF₃ substituentsin the para position would extend toward the deepest corner of thebinding site which would result in improved molecular contact. Forcompound LFM-A3, the C-F bond length (1.3 Å) is shorter than the C—Cland C—Br bond lengths in LFM-A2 and LFM-A1 (1.8-1.9 Å) and thus the p-Fgroup would not approach Lys⁷²¹ as closely. This weaker contact maycontribute to the poor K_(i) values for LFM-A3. The para substitutedcompounds appeared to approach Asp⁸³¹ of EGFR more closely compared toortho or meta substituted compounds. The result of this closer approachmay be some steric strain with Asp⁸³¹ which may force the residue torotate away from the inhibitor as was observed in docking results. Thisaction actually disrupts a hydrogen bond between Asp⁸³¹ and Asn⁸³⁰ whichcould cause a slight destabilization of the protein conformation in thisregion. This event is more likely to occur when larger para-substitutedgroups are involved, such as the para-Br of LFM-A1 and para-Cl ofLFM-A2. The calculations did not incorporate the energetic effects ofthis speculated protein destabilization. Therefore, the true K_(i)values for compounds containing a larger para-substituted group, such asLFM-A1 and LFM-A2, may be higher than the estimated K_(i) values shownin Table 2.

In these studies, the para substituted CF₃ was more active than them-CF₃ compound in terms of IC₅₀ inhibition values (IC₅₀=5.4 μM vs>100μM, Table 1), which is consistent with the modeling observation thatthis compound maintains closer contacts with the hinge region than them-CF₃ compound. As shown in FIG. 3, the meta substitution is stericallyless favorable for allowing a closer interaction with the hinge regionof EGF-R relative to para substituted LFM compounds, which maycontribute to a loss of hydrophobic contact in this region of thebinding site. This loss of hydrophobic contact would be reflected in alower calculated K_(i) value based on docking studies.

The superimposed docked positions of the three CF₃ substituted compounds(ortho, meta, and para) are shown in FIG. 4. The bulky orthosubstitution would prevent the ligands from having close contact withthe catalytic site and make it unlikely for this compound to showsignificant inhibitory activity. The para substituted compound, on theother hand, can maintain good contact with the hinge region of thereceptor and would likely be an effective inhibitor by our calculations.The inhibition values for meta substituted compounds were predicted tofall between those of the ortho and para substituted compounds. Thecalculated K_(i) values are consistent with their final docked positionswhich show that the m-CF₃ compound is located in-between the para andortho compounds. Docking studies also showed that the ring substituentsof most of the active compounds were predicted to be positioned betweenresidues Thr⁷⁶⁶ and Asp⁸³¹ of the EGFR.

The modeling studies suggested that LFM-A12 would exhibit potentEGFR-inhibitory activity. In order to test this hypothesis and validatethe predictive value of the described EGFR kinase domain homology model,LFM-A12, LFM, and 13 other LFM analogs listed in Table 2 were prepared.The three dimensional structures of 10 of these compounds weredetermined by single crystal X-ray diffraction. The crystal data,experimental parameters and refinement statistics are summarized inTable 1. All structures, except LFM-A2 (p-Cl), were found to beessentially planar in conformation, and all bond lengths and angles werein the expected range. For LFM-A2 the dihedral angle between thearomatic ring and side chain was close to 45°.

In all crystal structures except LFM-A 11 (m-F-), the ortho or metasubstituents were found to reside on the same side of the molecule asthe nitrile group. In LFM-A11, the phenyl ring is rotated so that themeta-F and nitrile groups are on opposite sides of the molecule. Themolecular coordinates of LFM and LFM analogs which were energy-minimizedand docked into the EGFR binding site in modeling studies adopted aconformation similar to that of their crystal structures.

The effects of a compound on EGFR tyrosine kinase and survival of humanbreast cancer cells can be determined using pharmacological models whichare well known to the art, or using the biological tests describedbelow.

EXAMPLE 4 Chemistry and Synthesis

All chemicals were purchased from Aldrich (Milwaukee, Wis.) and wereused without further purification. Except where noted, each reactionvessel was secured with a rubber septum, and the reaction was performedunder nitrogen atmosphere. ¹H spectra were obtained on a Varian Mercury300 instrument spectrometer (Palo Alto, Calif.) at ambient temperaturein the solvent specified. Melting points were determined using aFisher-Johns melting point apparatus and are uncorrected. FT-IR spectrawere recorded on a Nicolet Protege 460 spectrometer (Madison, Wis.).GC/MS spectra were obtained on a HP 6890 GC System (Palo Alto, Calif.)equipped with a HP 5973 Mass Selective Detector.

Scheme 1 shows the general synthetic scheme for the preparations of LFM,and LFM-A1-LFM-A14 (Kuo, E. A., et al. J. Med. Chem. 1996, 39, 4608;Sjogren, E. R., et al. J. Med. Chem. 1991, 34, 3295). Cyanoacetic acid 1was coupled with the REQUISITE substituted-aniline 2 in the presence ofdiisopropylcarbodiimide (DIC) to form 3. Compound 3 was treated with NaHand acylated with acetyl chloride to afford the final products.

General Synthetic Procedures

The general synthetic procedures are described in the literature. (Kuo,E. A., et al. J. Med. Chem. 1996, 39, 4608; Sjogren, E. R., et al. J.Med. Chem. 1991, 34, 3295). In general, 1,3-diisopropylcarbodiimide(1.75 g; 13.9 mmol) was added to a solution of cyanoacetic acid 1 (1.70g; 20.0 mmol) and the requisite substituted-aniline 2 (12.6 mmol) intetrahydrofuran (25 mL) at 0° C. The mixture was stirred for 12 hours atroom temperature.

The urea precipitate (reaction side product) was removed by filtrationand the filtrate was partitioned between ethyl acetate and 0.5 N HCl.The organic layer was sequentially washed with brine twice, dried overanhydrous Na₂SO₄ and concentrated by rotary-evaporation. The solidproduct was recrystallized from ethyl alcohol to give pure 3. Sodiumhydride (0.93 g; 60% in mineral oil; 23.2 mmol) was added slowly to thesolution of 3 (12.0 m mol) in tetrahydrofuran (40 mL) at 0° C. Afterstirring for 30 minutes at 0° C., the requsite acid chloride R₁COCl(1.04 g; 13.2 mmol) was added to the reaction mixture. The reaction wascontinued for another hour at room temperature and then was quenched bythe addition of acetic acid (2 mL). The mixture was poured into icewater (100 mL) containing 2.5 mL of hydrochloric acid to precipitate thecrude product, which was collected by filtration and washed with water.The final pure product was obtained by recrystallization.

Physical Data of Specific Compounds:

-   -   α-Cyano-β-hydroxy-β-methyl-N-[4-(trifluoromethyl)phenyl]-propenamide        (LFM). mp: 230-233° C.; IR (KBr): 3303, 2218, 1600 and 1555        cm⁻¹; ¹H NMR (DMSO-d₆): δ 11.01 (s, 1H, NH), 7.75 (d,J=8.4 Hz,        2H, ArH), 7.64 (d, J=8.4 Hz, 2H, ArH), 2.22 (s, 3H, CH₃); GC/MS        m/z 270 (M⁺), 161, 142, 111.    -   α-Cyano-β-hydroxy-β-methyl-N-(4-bromophenyl)propenamide        (LFM-A1). mp: 213-214° C.; IR (KBr): 3288, 2228, 1615, 1555        cm⁻¹; ¹H NMR (DMSO-d₆): δ 10.51 (s, 1H, NH), 7.49 (s, 4H, ArH),        2.25 (s, 3H, CH₃); MS (EI) m/z 282(M⁺+z), 280 (M⁺), 173, 171.    -   α-Cyano-β-hydroxy-β-methyl-N-(4-chlorophenyl)propenamide        (LFM-A2). mp: 209-211° C.; IR(KBr): 3298, 2223, 1598 and 1552        cm⁻¹; ¹H NMR (DMSO-d₆): δ 10.48 (s, 1H, NH), 7.54 (d, J=8.7 Hz,        2H, ArH), 7.45 (s br, 1H, OH), 7.36 (d, J=8.7 Hz, 2H, ArH), 2.25        (s, 3H, CH₃); MS (CI) m/z 236 (M⁺), 121, 127.    -   α-Cyano-β-hydroxy-β-methyl-N-(4-fluorophenyl)propenamide        (LFM-A3). mp: 165-166° C.; IR (KBr): 3298, 2218, 1610 and 1560        cm⁻¹; ¹H NMR (DMSO-d₆): δ 10.33 (s, 1H, NH), 7.80 (s br, 1H,        OH), 7.53 (m, 2H, ArH), 7.16 (m, 2H, ArH), 2.26 (s, 3H, CH₃);        GC/MS m/z 220 (M⁺), 111.    -   α-Cyano-β-hydroxy-β-methyl-N-[2-(trifluoromethyl)phenyl]-propenamide        (LFM-A4). mp: 61-63° C.; IR (KBr): 3435, 2209, 1619, 1952 and        1548 cm⁻¹; ¹H NMR (DMSO-d₆): δ 10.99 (s, 1H, NH), 8.03 (d, J=7.5        Hz, 1H, ArH), 7.67 (d, J=7.5 Hz, 1H ArH), 7.60 (dd, J=7.5, 7.5        Hz, 1H, ArH), 7.29 (dd, J=7.5, 7.5 Hz, 1H, ArH) 5.71 (s br, 1H        OH), 2.20 (s, 3H, CH₃); GC/MS m/z 270 (M⁺), 161, 141, 114.    -   α-Cyano-β-hydroxy-β-methyl-N-(2-bromophenyl)propenamide        (LFM-A5). mp: 98-100° C.; IR (KBr): 3351, 2214, 1609, 1585 and        1536 cm⁻¹; ¹H NMR (DMSO-d₆): δ 10.76 (s, 1H, NH), 8.06 (dd,        J=8.1, 1.5 Hz, 1H, ArH), 7.62 (dd, J=8.1, 1.5 Hz, 1H, ArH), 7.33        (m, 1H, ArH), 7.03 (m, 1H, ArH), 6.60 (s br, 1H, OH), 2.22 (s,        3H, CH₃); ); MS (EI) m/z 282(M⁺+z), 280 (M⁺), 173, 171.    -   α-Cyano-β-hydroxy-β-methyl-N-(2-chlorophenyl)propenamide        (LFM-A6). mp: 93-94° C.; IR (KBr): 3372, 2208, 1644, 1621 and        1587 cm⁻¹; ¹H NMR (DMSO-d₆): δ 10.96 (s, 1H, NH), 8.16 (d, J=8.1        Hz, 1H, ArH), 7.46 (dd, J=7.5, 1.5 Hz, 1H, ArH), 7.29 (m, 1H,        ArH), 7.08 (m, 1H, ArH), 2.22 (s, 3H, CH₃); MS (CI) m/z 236        (M⁺), 29, 127.    -   α-Cyano-β-hydroxy-β-methyl-N-(2-fluorophenyl)propenamide        (LFM-A7). mp: 118-119° C.; IR (KBr): 3409, 2212, 1613, 1591 and        1532 cm⁻¹; ¹H NMR (DMSO-d₆): δ 10.70 (s, 1H, NH), 7.91 (m, 1H,        ArH), 7.23 (M, IH, ArH), 7.13 (m, 2H, ArH), 7.10 (s br, 1H, OH),        2.22 (s, 3H, CH₃); GL/MS m/z 220 (M⁺), 111.    -   α-Cyano-β-hydroxy-β-methyl-N-[3-(trifluoromethyl)phenyl]-propenamide        (LFM-A8). mp: 182-184° C.; IR (KBr): 3303, 2221, 1619 and 1572        cm⁻¹; ¹H NMR (DMSO-d₆): δ 10.79 (s, 1H, NH), 8.05 (s br, 1H, OH)        8.04 (s, 1H, ArH), 7.75 (d, J=8.1 Hz, 1H, ArH), 7.53 (dd, J=8.1,        7.5 Hz, 1H, ArH), 7.42 (d, J=7.5 Hz, 1H, ArH), 2.24 (s, 3H,        CH₃); GL/MS m/z 270 (M⁺), 161.    -   α-Cyano-β-hydroxy-β-methyl-N-(3-bromophenyl)propenamide        (LFM-A9). mp: 184-185° C.; IR (KBr): 3303, 2228, 1610, 1595 and        1550 cm⁻¹; ¹H NMR (DMSO-d₆): δ 10.56 (s, 1H, NH), 7.89 (m, 1H,        ArH), 7.47 (m, 1H, ArH), 7.28 (m, 2H, ArH), 6.37 (s br, 1H, OH),        2.26 (s, 3H, CH₃); MS (EI) m/z 282 (M⁺+H, ⁸¹Br), 280 (M⁺+H,        ⁷⁹Br), 171, 173.    -   α-Cyano-β-hydroxy-β-methyl-N-(3-chlorophenyl)propenamide        (LFM-A10). mp: 184-187° C.; IR (KBr): 3293, 2221, 1610, 1595 and        1557 cm⁻¹; ¹H NMR (DMSO-d₆): δ 10.61 (s, 1H, NH), 7.76 (m, 1H,        ArH), 7.42 (m, 1H, ArH), 7.33 (M, 1H, ArH), 7.16 (m, 1H, ArH),        6.84 (S br, 1H, OH), 2.25 (s, 3H, CH₃); MS (CI) m/z 236 (M⁺).    -   α-Cyano-β-hydroxy-β-methyl-N-(3-fluorophenyl)propenamide        (LFM-A11). mp: 136-138° C.; IR (KBr): 3297, 2221, 1613, 1597 and        1567 cm⁻¹; ¹H NMR (DMSO-d₆): δ 10.54 (s, 1H, NH), 7.54 (m, 1H,        ArH), 7.33 (m, 2H, ArH), 6.93 (m, 1H, ArH), 2.27 (s, 3H, CH₃);        GL/MS m/z 220 (M⁺), 111.    -   α-Cyano-β-hydroxy-β-methyl-N-[4-(trifluoromethoxy)phenyl]-propenamide        (LFM-A12). mp: 182-183° C.; IR (KBr): 3308, 2213, 1625 and 1580        cm⁻¹; ¹H NMR (DMSO-d₆): δ 10.57 (s, 1H, NH), 7.90 (s br, 1H,        OH), 7.64 (d, J=8.7 Hz, 2H, ArH), 7.32 (d, J=8.7 Hz, 2H, ArH),        2.25 (s, 3H, CH₃); GL/MS m/z 286 (M⁺), 177, 108.    -   α-Cyano-β-hydroxy-β-methyl-N-(2,5-dibromophenyl)propenamide        (LFM-A13). mp: 148-150° C.; IR (KBr): 3353, 2211, 1648 and 1590        cm⁻¹; ¹H NMR (DMSO-d₆): δ 11.41 (s, 1H, NH), 8.57 (d, J=2.4 Hz,        1H, ArH), 7.55 (d, J=8.7 Hz, 1H, ArH), 7.14 (dd, J=8.7, 2.4 Hz,        1H, ArH), 7.10 (s br, 1H, OH), 2.17 (s, 3H, CH₃); MS (EI) m/z        362 (M⁺+4), 360 (M⁺+2), 358 (M⁺), 253, 251, 249, 150.    -   α-Cyano-β-hydroxy-β-methyl-N-(phenyl)propenamide (LFM-A14). mp:        134-135° C.; IR (KBr): 3281, 2214, 1605, 1579 and 1554 cm⁻¹; ¹H        NMR (DMSO-d₆): δ 10.33 (s, 1H, NH), 7.51 (d, J=7.5 Hz, 2H, ArH),        7.40 (s br, 1H, OH), 7.31 (dd, J=7.5, 7.5 Hz, 2H, ArH), 7.11 (m,        1H, ArH), 2.26 (s, 3H, CH₃); GL/MS m/z 202 (M⁺), 93.

EXAMPLE 5 Biological Tests

Immunoprecipitation of Recombinant Proteins from Insect Cells

Sf21 cells were infected with a baculovirus expression vector for BTK,JAK1, or JAK3, as previously reported (Vassilev, A., et al. J. Biol.Chem. 1998, 274, 1646-1656; Goodman, P. A., et al. J. Biol. Chem. 1998,273, 17742). Cells were harvested and lysed (10 mM Tris pH 7.6, 100 mMNaCl, 1% Nonidet P-40, 10% glycerol, 50 mM NaF, 100 mM Na₃VO₄, 50 mg/mlphenylmethylsulfonyl fluoride, 10 mg/ml aprotonin, 10 mg/ml leupeptin)and the kinases were immunoprecipitated from the lysates, as reported(Vassilev, A., et al. J. Biol. Chem. 1998, 274, 1646-1656).

Antibodies used for immunoprecipitations from insect cells are asfollows: Polyclonal rabbit anti-BTK serum (Mahajan, S., et al. Mol.Cell. Biol. 1995, 15, 5304), polyclonal rabbit anti-JAK1 (HR-785), cat#sc-277, rabbit polyclonal IgG affinity purified, 0.1 mg/ml, Santa CruzBiotechnology; and polyclonal rabbit anti-JAK3 (C-21, cat # sc-513,rabbit polyclonal IgG affinity purified, 0.2 mg/ml, Santa CruzBiotechnology). Kinase assays were performed following a 1 hour exposureof the immunoprecipitated tyrosine kinases to the test compounds, asdescribed in detail elsewhere (Mahajan, S., et al. Mol. Cell. Biol.1995, 15, 5304; Uckun, F. M., et al. Science 1996, 22, 1096). Theimmunoprecipitates were subjected to Western blot analysis as previouslydescribed (Vassilev, A., et al. J. Biol. Chem. 1998, in press).

Cell Lines, Reagents, and Biochemical Assays:

MDA-MB-231 (ATCC HTB-26) and MDA-MB-361 (ATCC HTB-27) are EGFR positivehuman breast cancer cell lines (Uckun, F. M., et al. Clin. Can. Res.1998, 4, 901). These cell lines were maintained in RPMI 1640 mediumsupplemented with 10% fetal bovine serum. For subculturing, medium wasremoved from the flasks containing a confluent layer of cells, and fresh0.25% trypsin was added for 1-2 min. Trypsin was removed and culturesincubated for 5-10 min. at 37° C. until cells detached. Fresh medium wasthen added, and cells were aspirated and dispensed into new flasks.COS-7 simian kidney cell line and HepG2 human hepatoma cell line wereobtained from ATCC.

Antibodies directed against BTK, JAK1, JAK3, and HCK have been describedpreviously (Vassilev, A., et al. J. Biol. Chem. 1998, in press; Goodman,P. A., et al. J. Biol. Chem. 1998, 273, 17742; Mahajan, S., et al. Mol.Cell. Biol. 1995, 15, 5304; Uckun, F. M., et al. Science 1996, 22,1096). Polyclonal antibodies to BTK were generated by immunization ofrabbits with glutathione S-transferase (GST) fusion proteins (PharmaciaBiotech Inc.) containing the first 150 amino acids of BTK. Themonoclonal anti-Fas antibody (F22120) was obtained from the TransductionLaboratories, Inc. (Lexington, Ky.).

Immunoprecipitations, immune-complex protein kinase assays, andimmunoblotting using the ECL chemiluminescence detection system(Amersham Life Sciences) were conducted as described previously(Vassilev, A., et al. J. Biol. Chem. 1998, in press; Goodman, P. A., etal. J. Biol. Chem. 1998, 273, 17742; Mahajan, S., et al. Mol. Cell.Biol. 1995, 15, 5304; Uckun, F. M., et al. Science 1996, 22, 1096).

Horse radish peroxidase-conjugated sheep anti-mouse, donkey anti-rabbitsecondary antibodies and ECL reagents were purchased from Amersham(Oakbrook, Ill.). For insulin receptor kinase (IRK) assays, HepG2 humanhepatoma cells grown to approximately 80% confluency were washed oncewith serum-free DMEM and starved for 3 hour at 37° in a CO₂ incubator.Subsequently, cells were stimulated with insulin (Eli Lilly, cat#CP-410;10 units/ml/10×10⁶ cells) for 10 minutes at room temperature.Following this IRK activation step, cells were washed once with serumfree medium, lysed in NP-40 buffer and IRK was immunoprecipitated fromthe lysates with an anti-IRb antibody (Santa Cruz, Cat.# sc-711,polyclonal IgG). Prior to performing the immunecomplex kinase assays,the beads were equilibrated with the kinase buffer (30 mM Hepes pH 7.4,30 mM NaCl, 8 mM MgCl2, 4 mM MnCl₂).

For HCK kinase assays, we used HCK-transfected COS-7 cells. The cloningand expression of HCK in COS-7 cells has been described previously(Saouaf, S. J., et al. J. Biol. Chem. 1995, 270, 27072). The pSV7c-HCKplasmid was transfected into 2×10⁶ COS-7 cells using Lipofectamine(GIBCO/BRL), and the cells were harvested 48 hours later. The cells werelysed in NP-40 buffer and HCK was immunoprecipitated from the whole celllysates with an anti-HCK antibody.

Immune-Complex Kinase Assays and Anti-Phosphotyrosine Immunoblotting:

For EGFR immune complex kinase assays, 24-hours after treatment withleflunomide analogs, MDA-MB-231 breast cancer cells were stimulated with20 ng/mL EGF for 5 minutes, lysed in 1% Nonidet-P-40 buffer, and celllysates were immunoprecipitated with an anti-EGFR antibody reactive withthe sequence Ala³⁵¹-Asp³⁶⁴ of the human EGFR (Upstate Biotechnology Inc.[UBI] Catalog # 05-104) (Uckun, F. M., et al. Clin. Can. Res. 1998, 4,901). EGFR immune complexes were examined for tyrosine phosphorylationby Western blot analysis, as described by Uckun, F. M., et al. Clin.Can. Res. 1998, 4, 901. All anti-phosphotyrosine Western blots weresubjected to densitometric scanning using the automated AMBIS system(Automated Microbiology System, Inc., San Diego, Calif.) and for eachtime point a % inhibition value was determined by comparing the densityratios of the tyrosine phosphorylated EGFR protein bands to those of thebaseline sample and using the formula: % Inhibition=100−100×[Density oftyrosine phosphorylated EGFR band]_(test sample)/[Density of tyrosinephosphorylated EGFR band]_(baseline control sample). The IC₅₀ valueswere determined using an Inplot program (Graphpad Software, Inc., SanDiego, Calif.).

In other experiments, MDA-MB-231 cells were stimulated with 10 ng/ml EGFprior to immunoprecipitation of the EGFR. EGFR immune complexes wereincubated for 1 hour at room temperature with various LFM analogs andtyrosine kinase assays were performed in the presence of [γ-³²P]-ATP, aspreviously described (Uckun, F. M., et al. Clin. Can. Res. 1998, 4, 901;Narla, R. K., et al. Clin. Can. Res 1998, 1405; Uckun, F. M., et al.Science 1996, 22, 1096; Uckun, F. M., et al. Science 1995, 267, 886).The kinase assay gels were analyzed both by autoradiography and usingthe BioRad Storage Phosphor Imager System (BioRad, Hercules, Calif.) forquantitative scanning.

In Vitro Treatment of Cells with LFM Compounds:

In order to determine the cytotoxic activity of LFM and its analogsagainst breast cancer cells, cells in alpha-MEM supplemented with 10%(v/v) fetal calf serum were treated with various concentrations of thecompounds for 24 hours at 37° C., washed twice in alpha-MEM, and thenused in either MTT assays or in vitro invasion assays, as describedhereinafter.

Cytotoxicity Assay:

The cytotoxicity of various compounds against human breast cancer celllines was analyzed using the MTT(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay(Boehringer Mannheim Corp., Indianapolis, Ind.). Uckun F. M., et al.Clin. Cancer Res. 1998, 4, 901-912. Exponentially growing breast cancercells were seeded into a 96-well plate at a density of 2.5×10⁴cells/well and incubated for 36 hours at 37° C. prior to drug exposure.On the day of treatment, culture medium was carefully aspirated from thewells and replaced with fresh medium containing the LFM analogs atconcentrations ranging from 2 to 250 μM. Triplicate wells were used foreach treatment. The cells were incubated with the various compounds for36 hours at 37° C. in a humidified 5% CO₂ atmosphere. To each well, 10μl of MTT (0.5 mg/ml final concentration) was added and the plates wereincubated at 37° C. for 4 hours to allow MTT to form formazan crystalsby reacting with metabolically active cells. The formazan crystals weresolubilized overnight at 37° C. in a solution containing 10% SDS in 0.01M HCl. The absorbence of each well was measured in a microplate reader(Labsystems) at 540 nm and a reference wavelength of 690 nm.

To translate the absorbance A₅₄₀ values into the number of live cells ineach well, the A₅₄₀ values were compared to those on standardA₅₄₀-versus-cell number curves generated for each cell line. The percentsurvival was calculated using the formula: % survival=Live cellnumber[test]/Live cell number [control]×100. The IC₅₀ values werecalculated by non-linear regression analysis using an Graphpad Prismsoftware version 2.0 (Graphpad Software, Inc., San Diego, Calif.).

Confocal Laser Scanning Microscopy:

Immunofluorescence was used to examine the morphologic features ofbreast cancer cells treated with LFM and its analogs. Before theexperiment, cells were trypsinized from rapidly growing tissue cultureflasks and seeded onto sterile 22 mm² coverslips in 6-well cultureplates. Cells on coverslips were returned to the incubator for 24 hoursprior to treatment. The following day, drugs were added from a stocksolution made in DMSO to a final concentration of 100 μM. Final DMSOconcentration was 0.1% in both test samples and controls. Cells werereturned to a 37° C. incubator for 24 hours before further processing.

At 24 hours, coverslips were fixed in −20° C. methanol for 15 minutesfollowed by a 15 minute incubation in phosphate buffered saline +0.1%Triton X-100(PBS-Tx). Coverslips were next incubated with a monoclonalantibody against α-tubulin (Sigma Chemical Co, St. Louis, Mo.) at adilution of 1:1000 for 40 minutes in a humidified chamber at 37° C.Coverslips were washed for 15 minutes in PBS-Tx followed by a 40 minincubation with a goat anti-mouse IgG antibody conjugated to FITC(Amersham Corp., Arlington Heights, Ill.). The coverslips were againrinsed in PBS-Tx and incubated with 5 μM TOTO-3 (Molecular Probes,Eugene Oreg.) for 20 minutes to label the nuclear DNA. Coverslips wereimmediately inverted onto slides in Vectashield (Vector Labs,Burlingame) to prevent photobleaching, sealed with nail varnish andstored at 4° C. Slides were examined using a Bio-Rad MRC-1024 LaserScanning Confocal Microscope mounted on a Nikon Eclipse E800 uprightmicroscope with high numerical aperture objectives. Digital data wasprocessed using Lasersharp (Bio-Rad, Hercules Calif.) and AdobePhotoshop softwares (Adobe Systems, Mountain View, Calif.) and printedon a Fuji Pictography thermal transfer printer (Fuji, Elmsford, N.Y.).

Apoptosis Assays:

Loose packing of membrane phospholipid head groups and cell shrinkageprecede DNA fragmentation in apoptotic cells, thereby providing MC540binding as an early marker for apoptosis (Uckun F. M., et al. Clin.Cancer Res. 1998, 4, 901-912; and Kuo, E. A. J. Med. Chem. 1996, 39,4608-4621). Plasma membrane permeability to propidium iodide (PI, Sigma)develops at a later stage of apoptosis (Uckun F. M., et al. Clin. CancerRes. 1998, 4, 901-912; and Kuo, E. A. J. Med. Chem. 1996, 39,4608-4621). MC540 binding and PI permeability were simultaneouslymeasured in breast cancer cells 24 hours after exposure to leflunomideanalogs, as described (Uckun F. M., et al. Clin. Cancer Res. 1998, 4,901-912. Stock solutions of MC540 and PI, each at 1 mg/mL, were passedthrough a 0.22 μm filter and stored at 4° C. in the dark. Shortly beforeanalysis, suspensions containing 1×10⁶ cells were suspended in 5 μg/mLMC540 and 10 μg/mL PI and kept in the dark at 4° C. Whole cells wereanalyzed with a FACS Calibur or FACS Vantage flow cytometer (BectonDickinson, San Jose, Calif.). All analyses were done using 488 nmexcitation from an argon laser. MC540 and PI emissions were split with a600 nm short pass dichroic mirror and a 575 nm band pass filter wasplaced in front of one photomultiplier tube to measure MC540 emissionand a 635 nm band pass filter was used for PI emission.

Clonogenic Assays:

After treatment with LFM analogs, cells were resuspended in clonogenicmedium consisting of alpha-MEM supplemented with 0.9% methylcellulose,30% fetal bovine serum, and 50 μM 2-mercaptoethanol. Cells were platedin duplicate Petri dishes at 100,000 cells/mL/dish and cultured in ahumidified 5% CO2 incubator for 7 days. Cancer cell colonies wereenumerated on a grid using an inverted phase microscope of high opticalresolution (Uckun F. M., et al. Clin. Cancer Res. 1998, 4, 901-912; andKuo). Results were expressed as % inhibition of clonogenic cells at aparticular concentration of the test agent using the formula: %Inhibition=(1-Mean # of colonies [Test]/Mean # of colonies[Control])×100. Furthermore, the dose survival curves were constructedusing the percent control survival (=Mean # of colonies[Test]/Mean # ofcolonies [Control]×100) results for each drug concentration as the datapoints and the IC50 values were calculated. The IC50 values weredetermined using an Prism Version II Inplot program (Graphpad Software,Inc., San Diego, Calif.).

Transfilter Cell Invasion Assays:

The in vitro invasiveness of MDA-MB-231 human breast cancer cells wasassayed using a previously published method, which employsMatrigel-coated Costar 24-well transwell cell culture chambers (“Boydenchambers”) with 8.0 μm pore polycarbonate filter inserts which have beendemonstrated to permit the migration of human cancer cells (Yoshida, D.,et al. Neurosurgery 1996, 39, 360). The chamber filters were coated with50 μg/ml of Matrigel matrix, incubated overnight at room temperatureunder a laminar flow hood and stored at 4° C.

On the day of the experiment, the coated inserts were rehydrated with0.5 ml serum-free DMEM containing 0.1% bovine serum albumin for 1-2hours. To study the effects of LFM and LFM-A12 on invasiveness ofMDA-MB-231 cells, exponentially growing cells were incubated with eachcompound at various concentrations ranging from 12.5 RM to 100 μM in0.1% DMSO overnight. The cells were trypsinized, washed twice withserum-free DMEM containing BSA, counted and resuspended at 1×10⁵cells/ml.

A 0.5 ml cell suspension containing 5×10⁴ cells in a serum-free DMEMcontaining LFM, LFM-A12, or vehicle was added to the Matrigel-coated andrehydrated filter inserts. Next, 750 μl of NIH fibroblast conditionedmedium was placed as a chemoattractant in 24-well plates and the insertswere placed in wells and incubated at 37° C. for 48 hours. After theincubation period, the filter inserts were removed, the medium wasdecanted off and the cells on the top side of the filter that did notmigrate were scraped off with a cotton-tipped applicator. The invasivecells that migrated to the lower side of the filter were fixed, stainedwith Hema-3 solutions and counted under microscope. No cells weredetected at the bottom of the Boyden chambers. Therefore, the number ofcells on the lower side of the filters accounted for all cells that hadmigrated through the filter. Five (5) to ten (10) random fields perfilter were counted to determine the mean (±SE) values for the invasivefraction. The invasive fractions of cells treated with LFM or LFM-A12were compared to those of vehicle (0.1% DMSO in PBS) treated controlcells and the percent inhibition of invasiveness was determined usingthe following formula: % Inhibition=100×(1-Invasive Fraction ofDrug-Treated Cells/Invasive Fraction of Control Cells).

Results

Specific Inhibition of the EGFR Tyrosine Kinase by LFM-A12:

The effects of LFM analogs on the enzymatic activity of the EGFR kinasein cell-free immune complex kinase assays were examined. As shown inFIG. 5A, a one hour incubation with LFM or LFM-A12 inhibited the EGFRtyrosine kinase in a dose-dependent fashion in anti-EGFRimmunoprecipitates from lysates of MDA-MB-231 human breast cancer cells.The IC₅₀ values for EGFR inhibition were 5.4 μM for LFM and 1.7 μM forLFM-A12 (FIG. 5A). In contrast, the IC₅₀ values for all other compoundswere >100 μM, except for LFM-A7 which was 74.5 μM.

The effects of LFM and LFM analogs on the enzymatic activity of the EGFRtyrosine kinase in breast cancer cells were also examined. After a 24hour exposure to LFM or LFM analogs, MDA-MB-231 cells were stimulatedwith EGF for 10 minutes, and EGFR immune complexes from whole celllysates were subjected to Western blot analysis with a polyclonalanti-phosphotyrosine antibody to measure the autophosphorylation of theEGFR. Treatment of MDA-MB-231 cells with LFM-A12 and, albeit to a lesserextent, with LFM resulted in decreased tyrosine phosphorylation of theEGFR after EGF stimulation. In contrast, none of the other LFM analogstested side-by-side were able to inhibit the EGFR kinase in cell-free(Table 2) or cellular (not shown) EGFR kinase inhibition assays. Takentogether, these experimental results are consistent with the generaltrend for most of the calculated K_(i) values shown in Table 2, therebyconfirming the predictive value of the constructed homology model of theEGFR kinase domain.

In MTT assays (Uckun, F. M., et al. Clin. Can. Res. 1998, 4, 901; Narla,R. K., et al. Clin. Can. Res 1998, 1405), LFM-A12 exhibited significantcytotoxicity against the MDA-MB-361 human breast cancer cell line withmean IC₅₀ value of 26.3 μM. By comparison, LFM was significantly lessactive against these breast cancer cells. The IC₅₀ value for the LFMcomposite dose survival curves was 190.5 μM for MDA-MB-361 cells.

The inhibitory effects of LFM and LFM-A12 on the EGFR tyrosine kinasewere specific in that they did not affect the enzymatic activity ofother protein tyrosine kinases, including receptor family tyrosinekinase IRK (FIG. 7B), Src family tyrosine kinase HCK (FIG. 7C), Januskinases JAK1 and JAK3 (FIGS. 7D-E), and Tec family tyrosine kinase BTK(FIG. 7F), at concentrations as high as 350 μM (Table 3).

TABLE 3 LFM-A12 interaction scores, calculated K_(i) values and measuredIC₅₀ values for the inhibition of several protein tyrosine kinases.

Lipo Ludi Ludi^(b) Inhibition Protein HB^(a) Score Score K_(i)(μM)IC₅₀(μM) EGFR 1 510 489 13 1.7 BTK 1 496 457 27 >175 IRK 1 451 41178 >350 HCK 0 510 385 142 >350 JAK1 1 387 347 340 >175 JAK3 0 402 2771710 >350 ^(a)HB = Number of hydrogen bonds between inhibitor andprotein. ^(b)Ludi K_(i) calculated based on the empirical score functionin Ludi program (Bohm, H. J. J. Comput. Aided. Mol. Des. 1992, 6, 593;and 36. Bohm, H. J. J. Comput. Aided Mol. Des. 1994, 8, 243). Cell-freetyrosine kinase inhibition assays were performed as described above andthe IC50 values were calculated from the LFM-A12 concentration-kinaseactivity curves.

Modeling studies were performed using the crystal structure coordinatesof HCK (Sicheri et al., 1997, Nature, 385:602-9) and IRK (Hubbard, 1997,the EMBO Journal, 16:5572-5581) and constructed homology models for thekinase domains of JAK1, JAK3 ((Sudbeck et al., 1999, Clin. Can. Res. (inpress)) and BTK (Mahajan et al., 1999, JBC, in press) to identifypossible causes for the observed selectiveity of LFM-A12 for the EGFRtyrosine kinase. While most of the catalytic site residues of the EGFRkinase domain were conserved realtive to other PTKs, we noted a fewspecific variatoins. EGFR residues Leu⁶⁹⁴, Val⁷⁰², Lys⁷²¹, and Ala⁷¹⁹are conserved in EGFR, HCK, FGFR and IRK. Residues Asn⁸¹⁸ and Asp⁸³¹(opposite to hinge) are converved in EGFR, HCK, FGFR, IRK, BTK, JAK1 andJAK3. Residues Cys⁷⁵¹ and Thr⁸³⁰ are specific for EGFR but vary in BTK(Val, Ser), JAK1 (Val, Gly), JAK3 (Val, Ala), IRK (Val, Gly), and HCK(Val, Ala). Residues Thr⁷⁶⁶ and Leu⁷⁶⁸ in the hinge region changes toMet and Leu in IRK, Met and Phe in JAK1, Met and Tyr in JAK3, and to Thrand Tyr in BTK. One region of the binding pocket contains Cys⁷⁷³ in EGFRand is therefore considerably more hydrophobic than the correspondingresidue of PDGFR (Asp), FGFR (Asn), JAK1 (Ser), HCK (Ser), and IRK(Asp).

LFM-A12 was docked into the kinase domains of IRK, HCK, JAK3, JAK1, BTKand EGFR. After energy minimization the compound maintained favorableclose contacts with the hinge region of each kinase although theorientation of LFM-A12 in the catalytic site was different for BTK andHCK as shown in FIG. 6A. When bindign with EGFR the inhibitor appearedto be sanwiched between four residus, Leu⁶⁹⁴ and Val⁷⁰² from above andLeu⁸²⁰ and Thr⁸³⁰ from below. The nitrile nitrogen of the ligand wasinvolved in hydrogen bonding with the amide NH of Met⁷⁶⁹. In addition,the para-substituted OCF₃ group on the lead compound appeared to formclose contacts between residues Thr⁷⁶⁶ and Asp⁸³¹ at positions A and C,respectively, of EGFR (FIG. 6B).

Table 3 shows the interaction scores, estimated K_(i) values, andmeasured IC₅₀ data for LFM-A12 with the different kinases. The dataindicated that the selectivity of LFM-A12 for EGFR likely results fromits molecular shape and from favorable interactions with unique EGFRresidues that are not present in the kinase domains of the other PTKs.Likewise, unfavorable interactions with unique residues of the otherPTKs that are not found in the EGFR kinase domain also contribute tothis selectivity. These residue differences are illustrated in FIGS. 6Aand 6B at positions A and B.

FIG. 6A shows the backbone of the EGFR catalytic site, the residuedifferences between EGFR (white) and other kinases, and the dockedposition of LFM-A12 (multi-color) at this site in BTK (peach) which isalso similar to the docked position in HCK (blue). FIG. 6B shows thedocked position of LFM-A12 (multi-color) in EGFR (white), which is alsosimilar to the docked position in JAK3 (pink) and IRK (green). Thedotted surface area in each figure represents the Connolly surface ofthe inhibitor LFM-A12.

The aromatic residue in BTK (Tyr), HCK (Phe), JAK1 (Phe), JAK3 (Tyr)(shown at position B in FIGS. 6A and 6B, is not as favorable forinteractions with the p-OCF₃ group of LFM-A12. The corresponding residuein the EGFR kinase domain is leucine (shown in white at position B inFIGS. 6A and 6B), which would not cause such unfavorable interactionswith LFM-A12. Also, for HCK there is a loss of hydrogen bondinginteraction with LFM-A12. Furthermore, JAK3, IRK (shown in FIG. 6B), andJAK1 (not shown) contain a methionine residue (at position A in FIG. 6B)which protrudes into the active site and could impair the closehydrophobic contact of LFM-A12 with the hinge region of the catalyticsite. The longer methionine residue in JAK3 and IRK does not complimentthe shape of LFM-A12 and may hinder its binding. As shown in FIG. 6B,the corresponding residue in the EGFR kinase domain is threonine(white); its relatively shorter side chain enables LFM-A12 (multicolor)to have a more favorable hydrophobic contact with the hinge region whichmay result in tighter binding to the EGFR binding site. For EGFR, themost active compound (LFM-A12) appears to be located between theresidues at positions A and C. Consequently, the estimated K_(i) valuefor the EGFR (13 μM) was lower than the K_(i) values for other PTKswhich ranged from 27 μM for BTK to 1710 μM for JAK3 (Table 3).

Confocal Imaging

The effects of LFM-A12 treatment on MDA-MB-231 cells were examined byconfocal laser scanning microscopy. Slides were examined using a Bio-RadMRC-1024 Laser Scanning Confocal Microscope mounted on a Nikon EclipseE800 upright microscope with high numerical aperture objectives. Digitaldata was processed using Lasersharp (Bio-Rad, Hercules Calif.) and AdobePhotoshop software (Adobe Systems, Mountain View, Calif.) and printed ona Pictography printer (Fuji, Elmsford, N.Y.). Bohm, H. J. J. Comput.Aided Mol. Des., 1994, 8, 243-256.

As shown in FIG. 8A, vehicle (DMSO) treated control cells were round andlarge with many well organized microtubules (green fluorescencesecondary to tubulin staining) in the cytoplasm. Nuclei (bluefluorescence secondary to TOTO-3 staining) were also round andhomogenous. In contrast, MDA-MB-231 cells treated with 100 μM LFM-A12for 24 hours were much smaller and had an abnormal shape with largecytoplasmic vacuoles (FIG. 8B). The microtubules of LFM-A12 treatedcells were fewer in number and they appeared less organized than thoseof DMSO treated controls. The nuclei (blue) of the LFM-A12 treated cellswere also smaller and misshapen. Unlike LFM-A12, 100 EM LFM-A3 did notaffect the morphology or microtubular organization of MDA-MB-231 cells(FIG. 8C).

Apoptosis Assays

The morphologic features of LFM-A12 treated MDA-MB-231 cells byimmunocytohemistry (i.e., shrinkage, nuclear condensation, and abnormalmicrotubular organization) suggested that these cells might beundergoing apoptosis. Therefore, to determine whether LFM-A12 couldtrigger apoptosis in breast cancer cells, a quantitative flow cytometricapoptosis detection assay was performed.

Loose packing of membrane phospholipid head groups and cell shrinkageprecede DNA fragmentation in apoptotic cells, thereby providing MC540binding as an early marker for apoptosis (Uckun, F. M. et al., Science,1995, 267, 886-891). MC540 binding and propidium iodide (PI)permeability of MDA-MB-231 breast cancer cells were simultaneouslymeasured before and after a 24 hour treatment with 100 μM or 500 μMLFM-A12. Whole cells were analyzed with a FACStar Plus flow cytometer(Becton Dickinson, San Jose, Calif.). Whereas less than 5% of MDA-MB-231cells showed apoptotic changes after DMSO treatment, a significantportion of cells underwent apoptosis within 24 hours after LFM-A12treatment (Apoptotic fraction [AF] with MC540⁺/PI⁺ advanced stageapoptosis: 54% at 100 μM and 85% at 500 μM) (FIG. 9). LFM, albeit to alesser extent, also induced apoptosis in MDA-MB-231 cells.

Clonogenic Assays

The anti-cancer activity of LFM and LFM-A12 against MDA-MB-361 andMDA-B-231 breast cancer cells was tested using in vitro clonogenicassays. As shown in Table 4, 24 hour treatment with LFM or LFM-A12inhibited the clonogenic growth of MDA-MB-361 cells as well asMDA-MB-231 cells in a dose-dependent fashion. At 100 μM, our leadcompound LFM-A12 killed 87.3% of clonogenic MDA-MB-361 cells and >99% ofclonogenic MDA-MB-231 cells.

TABLE 4 In Vitro Anti-Tumor Activity of LFM and LFM-A12 AgainstClonogenic Breast Cancer Cells Tumor Cell Colonies/10⁵ Cell LineTreatment Cells % Inhibition MDA-MB-361 None 1104 (924, 1284) 0 DMSO(0.1%) 1088 (872, 1304) 1.4 LFM 0.1 μM 803 (702, 904) 27.3 10 μM 535(386, 684) 51.5 100 μM 196 (128, 264) 82.3 LFM-A12 0.1 μM 746 (316,1276) 32.4 10 μM 440 (276, 604) 60.2 100 μM 140 (58, 222) 87.3MDA-MB-231 None 1150 (1096, 1204) 0 DMSO (0.1%) 953 (888, 1018) 17.1 LFM0.1 μM 964 (588, 1340) 16.2 10 μM 642 (572, 712) 44.2 100 μM 297 (170,424) 74.2 LFM-A12 0.1 μM 667 (454, 880) 42.0 10 μM 515 (420, 610) 55.2100 μM 0 >99Effects of LFM-A12 on MDA-MB-231 Breast Cancer Cell Invasion ThroughMatrigel Matrix:

Matrigel matrix is made up of growth factors and several extracellularmatrix (ECM) components, including collagens, laminin and proteoglycans.As shown in FIGS. 7A and 7B, MDA-MB-231 human breast cancer cells werehighly invasive in Matrigel-coated Boyden chamber (CON). LFM-At2inhibited the invasion of MDA-MB-231 cells through the Matrigel matrixin a dose-dependent fashion with an IC₅₀ value of 28.4 μM and it wasmore potent than LFM which had IC₅₀ value of 97.0 FM (FIGS. 7A and 7B).

EXAMPLE 6 Design of New Analogs

Exploring the Catalytic Site of EGFR for the Design of SpecificInhibitors

The binding volume of the EGFR catalytic site is much larger than thevolume occupied by the lead compound LFM-A12. Increasing the size of theligand is postulated to increase the contact area between the receptorand ligand and hence enhance binding.

Structural and chemical features of LFM analogs which aid binding to theEGFR catalytic site are described below and illustrated in FIGS. 10-11.Table 5 shows the residue differences at the ATP binding site betweenthe six PTK's: EGFR, Btk, Hck, Jak1, Jak3 and IR.

TABLE 5 Residue differences between EGFR, Btk, Hck, Jak1, Jak3 and IR atthe ATP binding pocket. No EGFR Btk Jak1 Jak3 IR Hck 1 Cys 751 Val ValVal Val Val 2 Leu 764 Ile Leu Leu Val Ile 3 Thr 766 Thr Met Met Met Thr4 Leu 768 Tyr Phe Tyr Leu Phe 5 Cys 773 Cys Ser Cys Asp Ser 6 Arg 817Arg Arg Arg Arg Ala 7 Thr 830 Ser Gly Ala Gly Ala

T830, C751, T766, L764, L768, C773, R817 are some selected residues ofthe EGFR ATP binding site. Some or all of these residues are differentfrom the ATP binding site of Btk, IR, Jak1, Jak3 and Hck. Amongst them,the residues Thr830 and Cys751 are specific for EGFR. These nonconservedresidues can be utilized for the design of more potent and selectiveinhibitors of EGFR. In particular, targeting these residues forinteraction with specific inhibitor moieties would impact the bindingand stability of the inhibitor in the binding pocket, and enhance thespecificity of the inhibitor for EGFR.

Listed in Table 6 are some groups which are proposed to aid binding tothe EGFR catalytic domain, for example by providing favorable groups forinteraction with the EGFR kinase active site.

TABLE 6 Substitutions on LFM analogs likely to increase the bindingaffinity for EGFR. No Targeting Effect Substitutions 1 Cys 751 wouldincrease the Hydrophobic groups reaching specificity for EGFR about 5.5Å down from para-O 2 Leu 764 would not interact with Small hydrophobicgroups Btk, IR and Hck approximately within 3 Å from para-O 3 Thr 766would not interact with Small hydrogen bonding groups Jak1, Jak3 and IRapproximately within 3 Å from ortho and meta positions of ring (facinghinge). 4 Leu 768 5 Cys 773 would not interact with Hydrophobic groupsreaching Jak1 and IR. about 3.0 Å down from OH group of ligand 6 Arg 817would not interact with Long chain charged group Hck stretching about7.0-8.0 Å from OH or O group of ligand. 7 Thr 830 would increase theHydrophobic groups reaching specificity for EGFR approximately 3.5 Ådown from ortho and meta positions of ring (opposite hinge).

Referring to FIG. 10A, the lead compound LFM-A12 is shown bound to theEGFR-kinase domain in the predicted binding mode. A second possiblebindign mode is shown in FIG. 10B. Our modeling studies indicate thatpara substituted compounds maintain good contact with the hinge regionfo the receptor and appear to be good inhibitors by our calculations.For the para substituted compounds, a second mode of binding may also bepossibly where the molecule is roated 180° such that the aromatic ringis near residues P770, F771, G772, and the chain is new residues L764,T766, D831 (FIG. 10B).

In order to increase the affinity and specificity of the existing ligang(LFM-Al2), more ineractions with the active site residues are desired.Substitutions at positions R₁ and R₆-R₇ of formula II are expected tolead to increased binding affinity at the catalytic site of EGFR. Thisexpectation is based on the observation that the catalytic site of theEGFR kinase domain is much larger (volumne of about 500 Å³) than thevolue occupied by our most potent compounds. Increasing the size of theligand, preferably to fill up to about ⅔ of the volume (about 400 Å³) ispredicted to increase the contact area between the receptor and ligandand thus enhance binding.

The designed compounds of formulae III-VI are expected to provide suchincreased contact with the receptor, have enhanced binding, and potentinhibitory activity.

Novel compounds designed to fit and interact with specific contactpoints of the EGFR-TK binding pocket have the following structuralformulae (III-VI):

where R₅ is H, NH₂, CH₃, OH, CF₃, or halo. Preferably, R₅ is not H, andhalo is Br or Cl.

where R₅ is H, NH₂, CH₃, OH, CF₃, or halo. Preferably, R₅ is not H, andhalo is F or Cl.

where R₄ is NH—CH₃ or OCH₃.

where R₁ is —CH₂—CH₂X and X is halo, preferably Cl or Br; or R₁ ^(—CH)₂CF₃; or R₁ is:

These compounds are synthesized as detailed in the following schemes,according to their type of modifications. By using the propersubstituted-aniline, the compounds shown as formulae III, IV, and V maybe synthesized by the same general synthetic pathway used above forExample 4 (Scheme 1). Synthetically, the entry for synthesizing thecompounds of formula VI is to use different acylating agent, acidchloride, in the last step of the synthetic pathway shown in Scheme 1.The following schemes illustrate the four general types ofmodifications, and general synthesis schemes for the desiredsubstituted-analines for the four types.

Scheme 3:

Synthesis of the Desired Substituted-Anilines for Type I Modification(Compounds of Formula III)

Scheme 3 illustrates a strategy for the synthesis of the desiredsubstituted-analines for Type-I modification. All the starting materialsused in Scheme 3 are commercially available.

Scheme 4:Synthesis of the Desired Substituted-Analines for Type II Modification(Compounds of Formula IV)

Scheme 4 is one approach to synthesizing all the neededsubstituted-analines for Type-II modification, e.g., the production ofcompounds of formula V. All the starting material used in Scheme 4 arecommercially available.

Scheme 5:Synthesis of the Desired Substituted-Analines for Type III Modification(Compounds of Formula V)

For type III modification, producing compounds of formula VI, thestarting substituted-analine for synthesizing the compound of R₃=—OCH,is commercially available. The starting material for synthesizing thecompound of R³=—NH—CH3 is shown in Scheme 5.

Scheme 6:The Desired Acid Chlorides for Type IV Modification (Compounds ofFormula VI)

For the type IV modification, to produce the compounds of formula VII,the desired acid chlorides, 4-morpholinecarbonyl chloride (4), mehtylchloroforunate, (5), methyl chlorothioformate (6), trifluoroacetylchloride (7), as shown in Scheme 6, are commercially available.

All publications, patents, and patent documents are incorporated byreference herein, as though individually incorporated by reference. Theinvention has been described with reference to various specific andpreferred embodiments and techniques. However, it should be understoodthat many variations and modifications may be made while remainingwithin the spirit and scope of the invention.

1. A method for inhibiting EGFR tyrosine kinase, the method comprisingcontacting the EGFR with a compound having the structural formula:


2. A method for inhibiting EGFR tyrosine kinase without inhibiting Srcfamily tyrosine kinases, Tec family tyrosine kinases, or Janus familytyrosine kinases, the method comprising contacting the EGFR with acompound having the structural formula:


3. A method of treating cancer cells that express EGFR, the methodcomprising contacting the cancer cells that express EGPR with a compoundhaving the structural formula: